Nucleotide sequences encoding a CS2 pilin protein

ABSTRACT

Provided herein is a nucleotide sequence encoding a CS2 pilin protein of enterotoxigenic Escherichia coli. Also provided herein are the nucleotide sequences of coding sequences linked to the CS2 coding sequence, which other coding sequences must be expressed to allow the synthesis and assembly of the CS2 pili on the cell surface.

This invention was made, at least in part, with funding from the United States National Institutes of Health. Accordingly, the United States Government may have certain rights in this invention.

FIELD OF THE INVENTION

The field of this invention is the area of vaccines and genes encoding bacterial virulence determinants, particularly those genes encoding pilin proteins and genes essential for the assembly of pili of enterotoxigenic Escherichia coli, most particularly the genes encoding CS2 pili and the linked genes essential for the assembly of CS2 pili.

BACKGROUND OF THE INVENTION

Diarrheal diseases are an important cause of death in infants and young children in developing countries. Diarrheal diseases are also an important cause of serious illness in the elderly or immune compromised adults, as well as in travelers to countries where sanitation is inadequate and where these diseases are endemic.

Enterotoxigenic Escherichia coli strains (ETEC) are a major cause of diarrheal disease. Accordingly, there is a need for a vaccine for preventing the diseases associated with these bacteria. Ideally, a vaccine generates a protective immune response in which it interferes with a very early step in the infectious process, for example, by preventing the attachment of the pathogenic bacterial cells to host tissue. In the case of ETEC, this attachment step is believed to be mediated by pili, which are long, proteinaceous structures extending out from the surface of the bacterial cells. The CS2 pili are believed to mediate attachment to and/or promote colonization of the human upper intestine.

Different human ETEC strains produce a number of serologically different types of pili. The pili differ in antigenic specificity and subunit (pilin) molecular weight. Among human ETEC strains there are CFA/I (colonization factor antigen 1), CS1 (coli surface antigen 1) and CS2 (coli surface antigen 2) Evans and Evans (1978) Infect. Immun. 21:638-647; Smyth (1982) J. Gen. Microbiol. 128:2081-2096!. The latter two were initially grouped together as CFA/II, a term that also includes the fibrillar CS3 surface structures Levine et al. (1984) Infect. Immun. 44:409-420!. These three pili types seem to form a family based on several kinds of data. Although present in different ETEC strains, they are all positively regulated by rns or a closely related gene Caron et al. (1989) Proc. Natl. Acad. Sci. USA 86:963-967; Caron et al. (1990) Infect. Immun. 58:3442-3444; Savelkoul et al. (1990) Microb. Pathogen. 8:91-99!. Second, the N-terminal amino acid sequences of the major pilin proteins are almost identical for these three pili types and for CS4 and PCF1066 Wolf et al. (1989) Infect. Immun. 57:164-173; Sommerfelt et al. (1992) Infect. Immun. 69:3799-3806!. Third, for CFA/I and CS1, the DNA sequences of the four genes required for pili expression show significant homology Hamers et al. (1989) Microbial Pathogenesis 6:297-309; Perez-Casal et al. (1990) Infect. Immun. 58:3594-3600; Froehlich et al. (1994) Molec. Microbiol. 12:387-401; Jordi et al. (1991) FEMS Microbiol. Lett. 80:265-270; Jordi et al. (1992) DNA Sequence 2:257-263!. Additionally, both CS1 and CFA/I are determined by plasmid-borne genes.

Only the genes encoding CS1 have been analyzed for function. The morphogenesis of CS1 pili appears to differ from that of other pili in Gram negative bacteria, for example, the pap pili of the E. coli strains associated with pyelonephritis or the common type 1 pili. Only four genes are needed to express CS1 pili in E. coli K12 Froehlich et al. (1994) supra!. The major pilin subunit, encoded by cooA, lacks the features of disulfide bonds and penultimate tyrosine residues common to many other pilins. Additionally, the CS1 and CFA/I proteins show no homology with any other known or predicted proteins in the databank Scott et al. (1992) Molec. Microbiol. 6:293-300; Froehlich et al. (1994) Molec. Microbiol. 12:387-401!. Thus, no typical chaperonins can be identified on the basis of sequence comparisons. Therefore, the pili on human ETEC strains are believed to belong to a unique family of pilins.

Mutations in any one of cooA, cooB, cooC and cooD result in an absence of pili. Only mutation in cooA prevents pilin synthesis (Scott et al. (1992) supra; Froehlich et al. (1994) supra!. There is no candidate for a pilus-associated adhesion other than the pilin protein itself.

There is a long felt need in the art for vaccines useful in protecting against diarrheal disease resulting from infection with ETEC, including CS2 ETEC. Because of the serological diversity in pili of ETEC, an ETEC vaccine advantageously includes antigenic determinants (epitopes) from more than one pilus type to be effective against more than one type of ETEC infection, or a single vaccine can include one or more conserved antigenic determinants.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a nucleotide sequence encoding a CS2 pilin from enterotoxigenic Escherichia coli. As specifically exemplified, the encoded CS2 pilin has a preprotein amino acid sequence as given in SEQ ID NO:3 from amino acids -23 to +147. The preprotein coding sequence is given in SEQ ID NO:1 from nucleotide 1255 to nucleotide 1764 (excluding the translation stop codon). The encoded mature CS2 pilin protein has a deduced amino acid sequence as given in SEQ ID NO:3 from amino acid 1 through amino acid 147. The exemplified coding sequence of the mature CS2 pilin is given in SEQ ID NO:1 from 1324 to nucleotide 1764 (excluding the translation stop codon).

It is an additional object of the invention to provide non-naturally occurring nucleic acid molecules for the recombinant production of CS2 pili. A further object of the invention is a non-naturally recombinant DNA molecule which carries all four genes of the CS2 pilin gene cluster. As specifically exemplified herein, the gene cluster is within the DNA sequence as given in SEQ ID NO:1, and in the presence of the rns gene product, this gene cluster directs the synthesis of CS2 pili.

It is a further object of this invention to provide non-naturally occurring nucleic acid molecules, i.e., recombinant polynucleotides (e.g., a recombinant DNA molecule) comprising a nucleotide sequence encoding a CS2 pilin, preferably having a mature amino acid sequence as given in SEQ ID NO:3 from amino acid 1 through amino acid 147 or having a precursor amino acid sequence as given in SEQ ID NO:3, amino acids -23 through 147. As specifically exemplified herein, the nucleotide sequence encoding a mature CS2 pilin is given in SEQ ID NO:1 nucleotides 1324 through 1767, or a precursor coding sequence SEQ ID NO:1 from nucleotides 1255 through 1767. The skilled artisan will understand that the amino acid sequence of the exemplified CS2 pilin protein can be used to identify and isolate additional, nonexemplified nucleotide sequences which will encode a protein of the same amino acid sequence as given in SEQ ID NO:3, from amino acid 1 through amino acid 147, or an amino acid sequence of greater than 90% identity thereto and having equivalent biological activity. When it is desired that the sequence encoding a CS2 pilin or other CS2 protein be expressed, then the skilled artisan will operably link transcription and translational control regulatory sequences to the coding sequences, with the choice of the regulatory sequences being determined by the host in which the coding sequence is to be expressed. With respect to a recombinant DNA molecule carrying a CS2 pilin coding sequence, the skilled artisan will choose a vector (such as a plasmid or a viral vector) which can be introduced into and which can replicate in the host cell. The host cell can be a bacterium, preferably Escherichia coli, or, alternatively, a yeast or mammalian cell.

In another embodiment, recombinant polynucleotides which encode one or more of the proteins Cot A, Cot B, Cot C, and Cot D, required for production of CS2 pili including, e.g., protein fusions or deletions, as well as expression systems are provided. Expression systems are defined as polynucleotides which, when transformed into an appropriate host cell, can express CS2 pilin or CS2 pili-related proteins. The recombinant polynucleotides possess a nucleotide sequence which is substantially similar to a natural CS2 pilin-encoding polynucleotide or a fragment thereof. Where CS2 pili assembly is desired, all four cot genes provided herein are expressed. Expression may be under the control of the promoter normally associated with these genes (and in the presence of a functional rns gene) or the cot gene cluster can be expressed under the regulatory control of a heterologous promoter. The preferred enteric bacterial host strain for pili production is a strain of E. coli (which contains and expresses the rns regulatory gene when expression is under the control of the cot operon promoter).

Further provided by the present invention are oligonucleotides and polynucleotides which are capable of hybridizing specifically, using standard conditions well understood by the skilled artisan, to CS2 ETEC genomic DNA (or to the cognate mRNA). It is understood that these nucleic acid molecules do not include those sequences within SEQ ID NO:1 which are homologous to IS1 or IS3 or which are homologous to the genetically unlinked rns gene. These CS2-specific sequences can also be used in the preparation of primers for use in the polymerase chain reaction (PCR) for the amplification of CS2 nucleic acid. Either hybridization or PCR can be adapted for use in the detection of CS2 ETEC bacteria in biological, food or environmental samples or in the diagnosis of disease caused by CS2 ETEC bacteria.

The polynucleotides include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases. DNA is preferred. Recombinant polynucleotides comprising sequences otherwise not naturally occurring are also provided by this invention, as are alterations of a wild type CS2 pilin or CS2 pili gene cluster sequence, including but not limited to deletion, insertion, substitution of one or more nucleotides or by fusion to other polynucleotide sequences, provided that such changes in the primary sequence of the CS2 pilin protein do not alter the epitope(s) capable of eliciting protective immunity.

The present invention also provides for fusion polypeptides comprising a CS2 pilin or a portion thereof. Homologous polypeptides may be fusions between two or more pilin protein sequences or between the sequences of a pilin and a related protein. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the proteins from which they are derived. Potential fusion partners include, but are not limited to, immunoglobulins, ubiquitin, bacterial β-galactosidase, trpE, protein A, β-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor Godowski et al. (1988) Science, 241, 812-816!. Fusion proteins are typically made by recombinant methods but may be chemically synthesized as well understood in the art.

Compositions and immunogenic preparations including, but not limited to, vaccines comprising recombinant CS2 pilin or CS2 pili derived from enterotoxigenic Escherichia coli or from a recombinant E. coli and a suitable carrier are provided by the present invention. Such vaccines are useful, for example, in immunizing a human against diarrheal disease resulting from infection by CS2 enterotoxigenic E. coli. The preparations comprise an immunogenic amount of a CS2 pilin protein or an immunogenic fragment thereof. Such vaccines can advantageously further comprise pilin proteins or antigenic determinants therefrom of one or more other serological types, including but not limited to, CS1, CS3, CS4, PCF1066 and/or CFA/I. By "immunogenic amount" is meant an amount capable of eliciting the production of antibodies, preferably conferring protective immunity directed against infection by enterotoxigenic E. coli with the same pilus types present in the vaccine with which an individual has been immunized.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations used herein for amino acids are standard in the art. The abbreviations for amino acid residues as used herein are as follows: A, Ala, alanine; V, Val, valine; L, Leu, leucine; I, Ile, isoleucine; P, Pro, proline; F, Phe, phenylalanine; W, Trp, tryptophan; M, Met, methionine; G, Gly, glycine; S, Ser, serine; T, Thr, threonine; C, Cys, cysteine; Y, Tyr, tyrosine; N, Asn, asparagine; Q, Gln, glutamine; D, Asp, aspartic acid; E, Glu, glutamic acid; K, Lys, lysine; R, Arg, arginine; and H, His, histidine.

CS2 pilin is the term given to the E. coli pilin protein having an amino acid sequence substantially as disclosed in SEQ ID NO:3. It is understood that "CS2 pilin" provides a specific immune reaction with CS2-specific antisera. "Cot A" and "CS2 pilin" are used interchangeably herein. The complete amino acid sequence of the exemplified CS2 pilin protein, including the signal peptide with which it is synthesized, is given in SEQ ID NO:3, from amino acid 1 through amino acid 147 (mature protein) and the amino acid sequence of the associated signal sequence is given in SEQ ID NO:3 from -23 to -1. In nature, these proteins are produced by enterotoxigenic E. coli and can be purified from cells or from culture supernatant or as recombinant expression products using the disclosure provided herein.

Recombinant CS2 pilin can be obtained by culturing host cells transformed with the recombinant polynucleotides comprising nucleotide sequences encoding CS2 pilin as described herein under conditions suitable to attain expression of the pilin-encoding sequence.

The purification of CS2 pili has been described in Smyth, C. J. (1982) J. Gen. Microbiol. 128:2081-2086; Smyth, C. J. (1984) FEMS Microbiol Lett. 21:51-57. Although the published purification is for pili removed from the surface of CS2 cells, CS2 pilin can also be purified from the recombinant host cells (preferably bacterial cells, most preferably E. coli) which express the pilin gene but do not assemble pili on the cell surface using modifications of the aforementioned purification procedures and/or other modifications readily apparent to one of ordinary skill in the art.

The cloned DNA carrying the CS2 gene cluster was identified using a CS2-specific antibody screen of the cosmid library made with total E. coli C91f-6 DNA. The functional expression of the CS2 pili was confirmed by a positive hemagglutination assay. The CS2 gene cluster was shown to be contained on a DNA fragment of about 5.7 kb and produces functional CS2 pili as measured by hemagglutination.

The DNA sequence of the 5.7 kb fragment was determined (See SEQ ID NO: 1). The CS2 gene cluster has 39% G+C, which is significantly lower than the 50% value for the E. coli genome as a whole. Analysis of that sequence revealed four open reading frames (ORFs) which appeared to be expressed under the control of one promoter region. The four ORFs were named cotA, cotB, cotC and cotD, with "cot" being a mnemonic for coli surface antigen two.

The first ORF (cotB) is at nucleotides 499-1212 in SEQ ID NO:1; it is 714 bases in length and is preceded by a potential ribosome binding site (AAGG) beginning at about 16 bp upstream of the ATG translation start site. The deduced amino acid sequence for this ORF is given in SEQ ID NO:2 and contains 238 amino acids. The amino acid sequence appears to contain an 18 amino acid signal sequence. Processing at the predicted signal peptide cleavage site generates a predicted protein of 24.8 kDa in the periplasm of the bacterial cell. The mature protein is encoded at nucleotides 553-1212, and the signal peptide is encoded as nucleotides 499-552 of SEQ ID NO:1.

The second ORF (cotA) is 510 nucleotides in length (corresponding to nucleotides 1255-1764 of SEQ ID NO:1) and encodes a predicted protein of 147 amino acids, preceded by a signal peptide of an additional 23 amino acids. The deduced amino acid sequence is presented in SEQ ID NO:3. The first 30 amino acids of the predicted mature protein correspond exactly to the N-terminal amino acid sequence previously determined Smyth et al. (1990) in Microbial Surface Components and Toxins in Relation to Pathogenesis, Ron and Rottern, Eds., Plenum Press, London!. The cotA open reading frame is preceded by a typical ribosome binding site sequence (TAAGG) which begins about 14 bp upstream of the ATG translation initiation codon. The mature protein is encoded at nucleotides 1324-1764, and the signal peptide is encoded at nucleotides 1255-1323 of SEQ ID NO:1.

The third coding sequence in the CS2 cluster, termed cotC herein, is 2598 nucleotides in length (nucleotides 1836-4433 of SEQ ID NO:1). There is a potential ribosome binding site sequence (AAG) starting at 14 bp upstream of the ATG translation start codon. This ORF is predicted to generate a mature protein of about 94.6 kDa after cleavage of a signal peptide of 26 amino acids (see SEQ ID NO:4). The mature protein is encoded at nucleotides 1914-4433, and the signal peptide is encoded at nucleotide 1836-1913 of SEQ ID NO:1.

The fourth ORF (cotD) is 1092 nucleotides in length (nucleotides 4451-5542 in SEQ ID NO:1). The potential translation initiation codon, associated with a candidate ribosome binding site sequence (GAGGT) about 10 bp upstream, is the less frequently used TTG codon. The deduced amino acid sequence (SEQ ID NO:5) generates a predicted mature protein of 364 amino acids (38.9 kDa) after cleavage of an 18 amino acid signal sequence. The mature protein is encoded at nucleotides 4505-5542, and the signal peptide is encoded at nucleotides 4451-4504 of SEQ ID NO:1.

A search of the combined Genbank databases using Blast software and the coding sequences from the CS2 gene cluster showed only significant homologies to the corresponding coding sequences of other human ETEC pili operons. There was significant homology of CotB to CooB (54% identical and 72% similar) and CfaA (52% identical and 71% similar), the first genes in the CS1 and CFa/I pili gene clusters, respectively Jordi et al. (1991) FEMS Microbiol. Lett. 80:265-270; Hamers et al. (1989) Microbial Pathogenesis 6:297-309!. The hydropathy profiles for the three proteins were similar, but the calculated pI for CotB is much lower (8.58) than for CooB (10.32) or CfaA (10.22). Table 1 presents a comparison of the amino acid sequences of the CooB (from CS1 ETEC), CotB (from CS2 ETEC) and CfaA (from Cfa/I ETEC) proteins (SEQ ID NO:12, SEQ ID NO:2, and SEQ ID NO:13, respectively).

Mature CotA, the CS2 pilin protein, was found to have significant amino acid homology to mature CooA (50% identical and 64% similar) and mature CfaB (51% identical and 68% similar) ETEC pilins. Furthermore, the N-terminal amino acid sequence of mature CS2 (CotA) pilin has significant homology with CS4 and PCF1066 pilins. The predicted hydrophobicity plots of the CotA, CooA and CfaB pilins are very similar, and the predicted pI values are all within the range between 4.86 and 5.5. Although there is some homology, these proteins appear serologically distinct, although McConnell et al (1989) FEMS Microbiol. Lett. 61:105-108 reports some weak cross-reactivity. Table 2 presents a comparison of the amino acid sequences of the CooA (CS1 pilin), CotB (CS2 pilin) and CfaA (Cfa/I pilin) proteins (SEQ ID NO:10, SEQ ID NO:3, and SEQ ID NO:11, respectively).

The predicted mature CotC protein shows significant amino acid sequence homology to the corresponding gene products of the third open reading frames of the CS1 and CfA/I gene clusters (CooC and CfaC, respectively) Froehlich et al. (1994) Molec. Microbiol. 12:387-401; Jordi et al. (1992) supra!, but the signal sequences of the preproteins exhibit little amino acid sequence homology. Mature CotC exhibits 58% identity and 73% similarity to CooC and 56% identity and 72% similarity to CfaC. The predicted pI values are 6.53, 5.36 and 6.88 for CotC, Cooc and CfaC, respectively, and the hydrophobicity plots for the three proteins are very similar. Table 3 presents a comparison of the amino acid sequences of the CooC (from CS1 ETEC), CotC (from CS2 ETEC) and CfaC (from Cfa/I ETEC) proteins (SEQ ID NO:14, SEQ ID NO:4, and SEQ ID NO:15, respectively).

The third genes of the three clusters have several properties in common with those of other outer membrane proteins for review, see Nikaido and Vaara (1985) Microbiol. Rev. 49:1-32; Jap and Walian (1990) Quart. Rev. Biophys. 23:367-403!. All three proteins have high predicted beta-sheet and low predicted alpha-helix contents as determined using the algorithm of Rost and Sander (1992) Nature 360:540; Rost and Sander (1993) J. Molec. Biol. 232:584-599; Sander and Schneider (1991) Proteins: Structure, Function and Genetics 9:56-68. These proteins have a relatively high content of charged amino acids and appear to lack extensive hydrophobic regions. Thus, it is concluded that CotC, CooC and CfaC are outer membrane proteins.

Limited homology was observed between CotC and other characterized outer membrane proteins including the Cfa1A protein of Yersinia pestis, which protein is involved in capsule biogenesis, a process deemed by some to be similar to pili formation Karlyshev et al. (1994) FEBS Lett. 297:77-80!. There is also limited homology to FanD and FimD, large outer membrane proteins required for the assembly of the K99 pili of animal ETEC strains and the type 1 pili of Salmonella typhimurium, which proteins in turn have some homology with a protein required for dissociation of subunits from a chaperonin. CooC has been shown to be required for the assembly of CS1 pili Froelich et al. (1994) supra!. Without wishing to be bound by any particular theory, it is postulated that CotC functions in the transport of growing pili through the outer membrane.

The only sequences similar to CotD in GenBank are the fourth genes of the CS1 and CFA/I gene clusters, CooD and CfaE Froehlich et al. (1994) supra; Jordi et al. (1992) supra!. Mature CotD is 52% identical and 67% similar to mature CooD and 50% identical and 68% similar to mature CfaE. The predicted hydrophobicities are very similar, but the predicted pI values of the mature proteins differ: 7.07 for CotD, 6.97 for CooD, and 9.22 for CfaE. Table 4 presents a comparison of the amino acid sequences of the CooD (from CS1 ETEC), CotD and CfaE (from Cfa/I ETEC) proteins (SEQ ID NO:16, SEQ ID NO:5, and SEQ ID NO:17, respectively).

The region upstream of the CS2 gene cluster coding sequences contains the promoter for at least the first gene (cotB) of the CS2 gene cluster and possibly includes regulatory elements. Part of the upstream region (nucleotides 1-369 of SEQ ID NO:1 is 99% homologous with the last 369 bases of the IS3 insertion sequence Timmerman and Tu (1985) Nucl. Acids Res. 13:2127-2139!. Directly following the IS3 homology is a region (nucleotides 369-401 of SEQ ID NO:1) which is 100% homologous with bases 73-41 of IS1 Ohtsubo and Ohtsubo (1978), Proc. Natl. Acad. Sci. USA 75:615-619!. There is a potential promoter (bases 422-451 of SEQ ID NO:1) upstream of the CotB open reading frame (which begins at nucleotide 499). This promoter is very likely to be utilized since both the -35 (gTGACA) and the -10 (TATcAT) regions match the consensus in 5 of 6 bases, and the spacing of 18 bases between the two regions is acceptable Pribnow (1979) in Biological Regulation and Development, Vol. I, R. F. Goldberger, Ed., Plenum Press, NY and London, pp. 219-277; Hawley and McClure (1983) Nucl. Acids Res. 11: 2237-2255!.

SEQ ID NO:1 extends 253 nucleotides beyond the stop codon of cotD. There are no open reading frames within this region, and a search of Genbank revealed no significant homology to any sequence therein.

Surprisingly, in an E. coli K12 genetic background, cotA cotB cloned under the regulatory control of the lac promoter appeared to produce relatively small amounts of CotA (CS2 pilin). Similarly, cotA alone also yielded small amounts of CS2 pilin protein even when expressed under the regulatory control of a heterologous promoter.

Oligonucleotides and polynucleotides which are capable of hybridizing specifically, using standard conditions well understood by the skilled artisan, to CS2 ETEC genomic DNA (or to the cognate mRNA) can be readily prepared using the CS2 sequence information provided herein (See SEQ ID NO:1 and the remainder of the present disclosure). It is understood that these nucleic acid molecules do not include those sequences within SEQ ID NO:1 which are homologous to IS3 or which are homologous to the genetically unlinked rns gene. These CS2-specific sequences can also be used in the preparation of primers for use in the polymerase chain reaction (PCR) for the amplification of CS2 nucleic acid. Either hybridization or PCR can be adapted for use in the detection of CS2 ETEC bacteria in biological, food or environmental samples or in the diagnosis of disease caused by CS2 ETEC bacteria. Where conditions for hybridization of the oligonucleotide or polynucleotide probe or primer, it is possible to obtain binding of the probe or primer to CS1 or Cfa/I pilin operon DNA. The skilled artisan can, using DNA sequence information provided herein and available in the prior art and with the choice of the proper positive and negative experimental controls, select the appropriate portion(s) of SEQ ID NO:1 to achieve the desired result of detection of or amplification of the selected target CS2, CS1, or Cfa/I nucleic acid. The appropriate adaptation of the technology allows the diagnosis of human ETEC disease caused by CS2, CS1 or Cfa/I bacteria (for example, using stool samples from a person having the symptoms of ETEC disease) as well as the detection of those bacteria in food, environmental or biological samples.

Because the gene products needed for CS2 and CS1 pili production share some homology, we determined whether they can complement each other for production of functional pili. Compatible plasmids were constructed to contain the A and B genes from one cluster and the C and D genes from the other. In these plasmids, the coo and cot genes were expressed from the lac promoter (see Example 2).

All complementation experiments were done in mutant derivatives of the ETEC-derived strain LMC10, which contains the genes for CS1 pili. Two mutants of LMC10 were used. The first, JEF100, expresses no coo gene products because it contains an omega insertion in the first gene in the cluster (cooB), which insertion is polar on the expression of downstream genes Scott et al. (1992) Mol. Microbiol. 6: 293-300!. JEF100 does not express pili, but the coo genes act in trans to complement this mutant for production of CS1 pili. For this complementation test, the cotA and B products were produced from pEU555, and the cooC and D products were produced from pEU478 in the JEF100 genetic background. Although JEF100 carrying either plasmid alone produces no visible pili, JEF100/pEU555/pEU478 was highly piliated, indicating the cotA and B gene products can interact with cooC and D gene products to assemble pili.

In the reciprocal experiment, a different LMC10 mutant was used. Strain FAK001 contains an omega insertion in cooC, so it expresses no CooC and little or no CooD. In the presence of a plasmid producing Rns, the positive regulator of the CS1 and CS2 genes, FAK001 expresses CooB and CooA Froehlich et al. (1994) supra!. FAK001/pEU2040 (a plasmid producing Rns) makes no visible pili. In the homologous complementation, pili are produced. Strain FAK001 carrying pEU2030 (which expresses Rns) and pEU478, which expresses CooC and D, is highly piliated. In the heterologous complementation, when pEU582, which encodes cotC and D, is present in FAK001/pEU2040, many pili are also produced. Thus the cooA and cooB gene products can also interact with the cotC and D gene products to form apparently normal pili.

Because cooC and cooD can substitute for cotC and cotD, and vice versa, strains which express both CS1 and CS1 can be constructed by introducing cotA and cotB into a genetic background which is Rns+, CooC+ and CooD+, and more preferably is deregulated for CS1 and/or CS2 pilin production. Similarly, the cooA and cooB genes can be introduced into a strain which is CotC+ CotD+ and is preferably deregulated for pilin production.

SEQ ID NO:1, nucleotides 1255-1764, presents an exemplified coding sequence for CS2 pilin with its associated 23 amino acid signal peptide. However, it is understood that there will be some variations in the amino acid sequences and encoding nucleic acid sequences for CS2 pilin from other CS2 ETEC strains. The ordinary skilled artisan can readily identify and isolate nonexemplified CS2 pilin-encoding sequences from other ETEC strains where there is at least 90% homology to the CS2 pilin coding sequence disclosed herein using this sequence taken with what is well known to the art. Those other sequences encode a pilin protein cross-reacting with and sharing antigenic determinants identical to those of the CS2 pilin exemplified herein.

It is understood by the skilled artisan that there can be limited numbers of amino acid substitutions in a protein without significantly affecting either function or antigenic cross reaction and specificity, and that nonexemplified CS2 pilin proteins can have some amino acid sequence divergence from the exemplified amino acid sequence (SEQ ID NO:13, amino acids 1-147). Such naturally occurring variants can be identified, e.g., by hybridization to the exemplified (mature) CS2 coding sequence (or a portion thereof capable of specific hybridization to CS2 pilin sequences) under conditions appropriate to detect at least about 90% nucleotide sequence homology, preferably about 95% homology. A requirement readily apparent to the skilled artisan is that a nonexemplified CS2 protein must be capable of generating protective immunity against ETEC of the CS2 serological type.

It is well known in the biological arts that certain amino acid substitutions can be made in protein sequences without affecting the function of the protein. Generally, conservative amino acids are tolerated without affecting protein function. Similar amino acids can be those that are similar in size and/or charge properties, for example, aspartate and glutamate, and isoleucine and valine are both pairs of similar amino acids. Similarity between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22, pages 345-352, which is incorporated by reference herein, provides frequency tables for amino acid substitutions which can be employed as a measure of amino acid similarity. Dayhoff et al.'s frequency tables are based on comparisons of amino acid sequences for proteins having the same function from a variety of different evolutionary sources.

The skilled artisan recognizes that other ETEC strains can have coding sequences for a pilin protein with the distinguishing characteristics of a CS2 pilin; those coding sequences may be identical to or synonymous with the exemplified coding sequence, or there may be some variation(s) in the encoded amino acid sequence. The distinguishing characteristics of CS2 pili or pilin are specific binding to CS2-specific antiserum and the ability to mediate mannose-resistant hemagglutination of bovine erythrocytes. It is noted that not all bovine erythrocytes are agglutinated by CS2 pili. Accordingly, it is understood by the skilled artisan that samples of bovine blood must be pre-tested with CS2-positive and CS2-negative controls prior to selection and use in such experiments. A CS2 pilin coding sequence from an ETEC strain other than C91f can be identified by, e.g., hybridization to a polynucleotide or an oligonucleotide having the whole or a portion of the exemplified coding sequence, under stringency conditions appropriate to detect a sequence of at least 90% homology, and where the expression product of said other coding sequence reacts specifically with the CS2-specific antiserum. In view of the similarity of the N-terminal amino acid sequences of ETEC pilin proteins (i.e., for CS1, CS2, CS4, PCF1066 and CFA/I pilins) the skilled artisan understands that a CS2-specific probe is directed to a portion of the coding sequence other than the 5' end of the pilin coding sequence. Similarly, it is understood that the portions of the CS2 gene cluster nucleotide sequence which have strong homology to a noncoding region of the unlinked rns locus or to IS3 or IS1 are also not appropriate to use as a hybridization probe for CS2 pilin coding sequences.

A polynucleotide or fragment thereof is "substantially homologous" (or "substantially similar") to another polynucleotide if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another polynucleotide, there is nucleotide sequence identity for approximately 80% of the nucleotide bases, usually approximately 90%, more preferably about 95% to 100% of the nucleotide bases.

Alternatively, substantial homology (or similarity) exists when a polynucleotide or fragment thereof will hybridize to another polynucleotide under selective hybridization conditions. Selectivity of hybridization exists under well known experimental conditions which allow one to distinguish the target polynucleotide of interest from other polynucleotides. Typically, selective hybridization can be effected when there is approximately 75% similarity over a stretch of about 14 nucleotides, preferably approximately 65%, more preferably approximately 75%, and most preferably approximately 90%. See Kanehisa (1984) Nucl. Acids Res. 12:203-213. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of about 17 to 20 nucleotides, and preferably about 36 or more nucleotides.

The hybridization of polynucleotides is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing polynucleotides, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and preferably in excess of 45° C. Stringent salt conditions will ordinarily be less than 1M, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter (Wetmur and Davidson (1968) J. Mol. Biol. 31, 349-370).

An "isolated" or "substantially pure" polynucleotide is a polynucleotide which is substantially separated from other polynucleotide sequences which naturally accompany a native CS2 sequence. The term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, chemically synthesized analogues and analogues biologically synthesized by heterologous systems.

A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide of a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

A nucleotide sequence is operably linked when it is placed into a functional relationship with another nucleotide sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Generally, operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, it is well known that certain genetic elements, such as transcription activators, may be operably linked even at a distance, i.e., even if not contiguous.

The term "recombinant" polynucleotide refers to a polynucleotide which is made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.

Polynucleotide probes include an isolated polynucleotide attached to a label or reporter molecule and may be used to identify and isolate nonexemplified CS2 pilin coding sequences. Probes comprising synthetic oligonucleotides or other polynucleotides may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or be chemically synthesized. Polynucleotide probes may be labelled by any of the methods known in the art, e.g., random hexamer labeling, nick translation, or the Klenow fill-in reaction.

Large amounts of the polynucleotides may be produced by replication in a suitable host cell. Natural or synthetic DNA fragments coding for a CS2 pilin or a fragment thereof will be incorporated into recombinant polynucleotide constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the construct will be suitable for replication in a unicellular host, such as yeast or bacteria, but a multicellular eukaryotic host may also be appropriate, with or without integration within the genome of the host cells. Commonly used prokaryotic hosts include strains of Escherichia coli, although other prokaryotes, such as Salmonella, Shigella, Vibrio or Bacillus subtilis may also be used. Mammalian or other eukaryotic host cells include yeast, filamentous fungi, plant, insect, amphibian and avian species. Such factors as ease of manipulation, degree and control of protein expression, ease of purification of expressed proteins away from cellular contaminants or other factors may determine the choice of the host cell.

The polynucleotides may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers (1981) Tetra. Letts. 22: 1859-1862 or the triester method according to Matteuci et al. (1981) J. Am. Chem. Soc. 103: 3185, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system (i.e., vector) recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides from the CS2 pilin coding sequence or from secreted polypeptides of the same or related species, are included, where appropriate, to allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989) vide infra; Ausubel et al. (Eds.) (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York; and Metzger et al. (1988) Nature 334: 31-36. Many useful vectors for expression in bacteria, yeast, mammalian, insect, plant or other cells are well known in the art and may be obtained from such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. In addition, the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences, see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, N.Y. (1983). While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.

Expression and cloning vectors will likely contain a selectable marker, that is, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. Although such a marker gene may be carried on another polynucleotide sequence co-introduced into the host cell, it is most often contained on the cloning vector. Only those host cells into which the marker gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g., ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper selectable marker will depend on the host cell; appropriate markers for different hosts are known in the art.

The recombinant vectors containing the CS2 coding sequence of interest can be introduced into the host cell by any of a number of appropriate means, including conjugation, electroporation, transformation or transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and transfection or infection (where the vector is an infectious agent, such as a viral or retroviral genome). The choice of such means will often depend on the host cell. Large quantities of the polynucleotides and polypeptides of the present invention may be prepared by transforming suitable prokaryotic or eukaryotic host cells with CS2 pilin-encoding polynucleotides of the present invention in compatible vectors or other expression vehicles and culturing such transformed host cells under conditions suitable to attain expression of the CS2 pilin-encoding sequence. The CS2 pilin may then be recovered from the host cell and purified.

The coding sequence for the mature CS2 pilin (CotA) is expressed after PCR site-directed mutagenesis and cloning into an expression vector suitable for use in E. coli in which the genes encoding Rns and preferably CotB, CotC or CooC and CotD or CooD are also expressed. Advantageously, the coding sequence also includes the information for a signal peptide. The signal peptide is removed during synthesis and localization by the cellular membrane proteins of the recombinant host cell. Exemplary expression vectors for E. coli and other host cells are given, for example, in Sambrook et al. (1989), vide infra, and in Pouwels et al. (Eds.) (1986) Cloning Vectors, Elsevier Science Publishers, Amsterdam, the Netherlands.

When it is desired to eliminate leader sequences and precursor sequences at the 5' side of the coding sequence, a combination of restriction endonuclease cutting and site-directed mutagenesis via PCR using an oligonucleotide containing a desired restriction site for cloning (one not present in coding sequence), a ribosome binding site, an translation initiation codon (ATG) and the codons for the first amino acids of the mature CS2 pilin. The oligonucleotide for site-directed mutagenesis at the 3' end of the coding sequence for CS2 pilin includes nucleotides encoding the carboxy-terminal amino acids of CS2 pilin, a translation termination codon (TAA, TGA or TAG), and a second suitable restriction endonuclease recognition site not present in the remainder of the DNA sequence to be inserted into the expression vector. The site-directed mutagenesis strategy is similar to that of Boone et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2800-2804, as modified for use with PCR.

In another embodiment, polyclonal and/or monoclonal antibodies capable of specifically binding to CS2 pilin or fragments thereof are provided. The term antibody is used to refer both to a homogenous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Monoclonal or polyclonal antibodies specifically reacting with the CS2 pilin may be made by methods known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, New York; and Ausubel et al. (1987) supra. Also, recombinant immunoglobulins may be produced by methods known in the art, including but not limited to the methods described in U.S. Pat. No. 4,816,567, incorporated by reference herein. Monoclonal antibodies with affinities of 10⁸ M⁻¹, preferably 10⁹ to 10¹⁰ or more are preferred.

Antibodies generated against CS2 peptide antigen and specific for CS2 pilin are useful, for example, as probes for screening DNA expression libraries or for detecting the presence of ETEC expressing CS2 pili in a test sample. Hydrophilic regions of the CS2 pilin can be identified by the skilled artisan See, e.g., Antibodies: A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.!, and peptide antigens can be synthesized and conjugated to a suitable carrier protein (e.g., bovine serum albumin or keyhole limpet hemocyanin) for use in vaccines or in raising antibody(ies) specific for CS2-expressing ETEC. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal. Suitable labels include but are not limited to radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. United States Patents describing the use of such labels include but are not limited to U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Antibodies specific for CS2 pilin and/or pili, which are capable of inhibiting adherence of CS2-expressing ETEC cells to host tissue, are useful in preventing diarrheal disease resulting from CS2 ETEC infection. Such antibodies can be obtained by the methods described above.

Compositions and immunogenic preparations including vaccine compositions comprising substantially purified recombinant CS2 pili or CS2 pilin protein and a suitable carrier therefor are provided. Alternatively, hydrophilic regions of the CS2 pilin can be identified by the skilled artisan, and peptide antigens can be synthesized and conjugated to a suitable carrier protein (e.g., bovine serum albumin or keyhole limpet hemocyanin) for use in vaccines or in raising antibody(ies) specific for CS2-expressing ETEC.

Alternatively, a suitable recombinant vector or recombinant host bacterium (such as a suitable Vaccinia virus or Mycobacterium) which carries an expressible gene encoding CS2 pilin or the CS2 pilin operon can be incorporated into a composition for immunizing a human. The coding sequence for at least one of CotA, CotB, CotC and CotD, preferably CotA (CS2 pilin) and optionally all four, can be incorporated into a recombinant nucleic acid molecule for use in a DNA vaccine preparation, termed a vaccine DNA preparation herein. Such a recombinant nucleic acid molecule (e.g., DNA) contains an origin of replication functional in human cells (e.g., an SV40 origin) and the coding sequence(s) is (are) operably linked to transcription regulatory elements such that when the recombinant DNA molecule is introduced into human cells, expression of the coding sequence(s) results. Optionally, there can be an origin of replication functional in a bacterium such as E. coli to facilitate cloning and amplification. When the vaccine DNA preparation is introduced into human cells in vivo, the coding sequence(s) is (are) expressed with the result that an immune response is raised to the protein expression product, and the vaccinated human develops immunity against CS2 ETEC infection and disease. The vaccine DNA preparation is formulated as known to the art, and the vaccine recombinant DNA molecules can be introduced into human cells in vivo or ex vivo. Ballistic transformation, liposome-mediated transformation, transfection, injection or any other means known to the art can be used to introduce the vaccine DNA molecules into human cells.

Immunogenic compositions are those which result in specific antibody production when injected into a human or an animal. Such immunogenic compositions are useful, for example, in immunizing a human against infection by CS2 ETEC. The immunogenic preparations comprise, or result in the expression of an immunogenic amount of one or more ETEC pilin or an immunogenic fragment(s) or subunit(s) thereof. Such immunogenic compositions may comprise one or more ETEC pilins, or in combination with another protein or other immunogen. By "immunogenic amount" is meant an amount capable of eliciting the production of antibodies directed against ETEC pilin, including but not limited to CS2 pilin in an individual to which the vaccine has been administered. Vaccines or immunogenic compositions comprising CS2 pili or pilin may also advantageously comprise other ETEC antigenic determinants, for example, pili or pilins of CS1, CS3, CS4, and Cfa and/or other serological types.

Immunogenic carriers may be used to enhance the immunogenicity of the CS2 pili or pilin. Such carriers include but are not limited to proteins and polysaccharides, liposomes, and bacterial cells and membranes. Protein carriers may be joined to the pilin sequences to form fusion proteins by recombinant or synthetic means or by chemical coupling. Useful carriers and means of coupling such carriers to polypeptide antigens are known in the art. The art knows how to administer immunogenic compositions so as to generate protective immunity on the mucosal surfaces of the gastrointestinal tract, where immunity specific for ETEC, and particularly CS2 type ETEC, is most helpful.

The immunogenic compositions may be formulated by any of the means known in the art. Such vaccines are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution or suspension in liquid prior to injection may also be prepared. The preparation may also, for example, be emulsified, or the protein encapsulated in liposomes.

The active immunogenic ingredients are often mixed with excipients or carriers which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. The concentration of the immunogenic polypeptide in injectable formulations is usually in the range of 0.2 to 5 mg/ml.

In addition, if desired, the vaccines may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE); and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogen resulting from administration of the immunogen in vaccines which are also comprised of the various adjuvants. Such additional formulations and modes of administration are known in the art and can also be used.

CS2 pilin or pili and/or fragments thereof may be formulated into immunogenic compositions as neutral or salt forms. Pharmaceutically acceptable salts include but are not limited to the acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic acids, e.g., acetic, oxalic, tartaric, or maleic acid. Salts formed with the free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.

The immunogenic CS2 pilin or pili (or peptide antigens thereof) compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of about 100 to 1,000 μg of protein per dose, more generally in the range of about 5 to 500 μg of protein per dose, depends on the subject to be treated, the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician and may be peculiar to each individual, but such a determination is within the skill of such a practitioner.

The vaccine or other immunogenic composition may be given in a single dose or multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as required to maintain and or reinforce the immune response, e.g., at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months.

All references cited herein are hereby incorporated by reference in their entirety.

Except as noted hereafter, standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those well known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.

The foregoing discussion and the following examples illustrate but are not intended to limit the invention. The skilled artisan will understand that alternative methods may be used to implement the invention.

EXAMPLES Example 1

Bacterial Strains and Plasmids

ETEC strain C91f-6 is a spontaneous CS2 pilus-deficient derivative of the wild-type ETEC strain C91f, which expresses CS2 Smyth (1982) J. Gen. Microbiol. 128:2081-2096; Smyth (1984) FEMS Microbiol. Lett. 21:51-57!. Strain LMC10 is a lac deletion, restriction-deficient derivative of the ETEC-derived C921b-2 strain Caron et al. (1989) supra!. Strain JEF100 is a derivative of LMC10 which contains the cooB1 allele in place of the wild type cooB Scott et al. (1992) Mol. Microbiol. 6:293-300!. The cooB1 allele contains an insertion of a kanamycin resistance omega fragment; the insertion mutation has polar effects, i.e., it inhibits the transcription and translation of downstream cistrons. Strain FAK001 is a LMC10 derivative in which there is a tetracycline resistance omega fragment inserted into cooC Froelich et al. (1994) Mol. Microbiol. 12:387-401!.

E. coli K-12 strains MC4100 and DH5alpha Casadaban, M. (1976) J. Mol. Biol. 104:557-566; Sambrook et al. (1989) supra! are used as host strains for cloning.

For most experiments bacteria are grown in Luria-Bertani (LB) broth as described Scott (1974) Virology 62:344-349!. For hemagglutination assays and for electron microscopy, bacteria are grown on CFA agar Evans et al. (1977) Infect. Immun. 18:330-337. Antibiotic supplementation, where necessary, is with ampicillin (50 μg/ml), chloramphenicol (40 μg/ml), kanamycin (40 μg/ml) or tetracycline (10 μg/ml).

The cosmid vector pHC79, which confers resistance to ampicillin Collins and Hohn (1980) Gene 11:291-298!, is used for the cloning of the genomic sequences containing the CS2 gene cluster. The high copy number vector pUC19, which also carries a gene conferring resistance to ampicillin Messing and Vieira (1982) Gene 10:269-276!, and the low copy number pSC101-based vector pHSG576, which confers resistance to chloramphenicol Takeshita et al. (1987) Gene 61:63-74!, are used for subcloning. The rns-containing plasmids used in these experiments are pEU2030 (rns cloned into pUC18) Caron et al. (1989) Proc. Natl. Acad. Sci. USA 86:963-967!, pEU2040 with rns cloned into pHSG576 Perez-Casal et al. (1990) Infect. Immun. 58:3594-3600! and pEU2021 with rns cloned into pBR322 Caron et al. (1989) supra!. Plasmid pEU478 contains cooC and cooD cloned under the regulatory control of the lac promoter in pHSG576 Froelich et al. (1994) supra!.

For CS2 expression, E. coli cells are grown at temperatures over 25° C., typically at 37° C.

Example 2

Immunological Techniques and Reagents and Hemagglutination Assays

Anti-CS2 polyclonal antibodies are prepared in rabbits as described by Scott et al. (1992) Molec. Microbiol. 6:293-300. C91f-6(pEU2021) cells are used as antigen for immunizing the rabbits. Antibodies which are not specific for the CS2 pili are removed by adsorption with sonicated cells of E. coli strains MC4100 and LMC10.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 12.5% gels is performed as in Laemmli (1970) Nature 227: 680-685. After SDS-PAGE, protein bands are transferred to nitrocellulose membranes, and immunoblotting is done using CS2-specific antiserum according to the protocol of Perez-Casal et al. (1989) Infect. Immun. 58:3594-3600. Total protein patterns are visualized with Coomassie blue staining or by the silver staining method See, e.g., Merril et al. (1979) Proc. Natl. Acad. Sci USA 76, 4335-4340!.

Hemagglutination assays using selected bovine erythrocytes are carried out in the presence of 100 mM mannose as described by Caron et al. (1989) Proc. Natl. Acad. Sci. USA 86:963-967. Not all bovine erythrocytes are agglutinated by CS2 pili. Accordingly, samples of bovine blood must pe pre-tested with CS2-positive and CS2-negative controls prior to selection and use in CS2 hemagglutination experiments. Equal volumes of 3% suspension of washed bovine erythrocytes and pelleted bacterial cells (grown overnight at 37° C. in LB are mixed and observed for hemagglutination after 30 minutes. A positive hemagglutination reaction is indicative of the expression of CS2 pili.

Example 3

Construction and Screening of the Total DNA library

A library is constructed using E. coli C91f-6 total DNA partially digested with Sau3A to give fragments of about 30-45 kb in size and BamHI-cut pHC79 cosmid DNA. After ligation the recombinant DNA molecules are packaged into lambda particles using a lambda packaging extract system (Gigapack, Stratagene, La Jolla, Calif.). Then the packaged recombinant cosmid particles are used to transduce E. coli DH5α containing pEU2040. Three of 452 ampicillin-resistant, chloramphenicol-resistant colonies react positively when tested by colony immunoblot analysis with CS2-specific antiserum. One isolate which is confirmed by Western blotting to contain CS2 antigen in heat extracts is studied further. This isolate contains a CS2-positive plasmid called pEU5006. After digestion of pEU5006 with PstI and EcoRV, a 5.7 kb fragment is isolated. This fragment is then ligated into pUC19 (which had been cut with PstI and SmaI) to yield pEU588. Said 5.7 kbp fragment has the nucleotide sequence of SEQ ID NO:1.

The following plasmids effect the expression of the CS2 genes under the regulatory control of the lac promoter of the vector. A 6.0 kbp ClaI/EcoRI fragment of PEU588 is made bluntended and ligated to SmaI-cut pUC19; this fragment contains the cotA and cotB genes. The EcoRI site of the fragment is about nucleotide 2753 of SEQ ID NO:1. pEU582 (containing cotC and cotD) is constructed by inserting a 4.0 PvuII fragment (extending from nucleotide 1642 to nucleotide 5591 of SEQ ID NO:1) into SmaI-cut pUC19. The presence and orientations of all inserts are confirmed by restriction endonuclease analysis.

Standard protocols for DNA library screening, plasmid phage purification, agarose gel electrophoresis and plasmid cloning were employed Maniatis et al. (1982), supra!.

Example 4

DNA Sequencing and Sequence Analysis

Double-stranded DNA is subcloned and sequenced by the dideoxy chain termination method Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467!. Oligonucleotide primers for sequencing reactions are synthesized by the phosphoramidite method with an Applied Biosystems model 394 automated DNA Synthesizer (Applied Biosystems, Foster City, Calif.).

Example 5

Electron Microscopy

Where it is desired to examine bacterial cells for the presence of pili, bacterial cells are grown at 37° C. on CFA agar supplemented with antibiotics to select for plasmid maintenance as necessary. A small sample of cells is suspended in 25 μl of a solution containing 1 Mm Tris-HCl (pH 7.5), 10 mM MgCl₂. The droplet of suspension is held for 60 minutes at room temperature. 10 μl of the sample is then diluted into 15 μl of the same Tris MgCl₂ solution, and formvar-coated carbon grids (200 mesh) are floated on the surface of the diluted cell mixture for 30 minutes. Excess liquid is absorbed from each grid using a paper wick, and each grid is then stained with ammonium phosphotungstate (pH 7.0). The grids are examined with a transmission electron microscope (Phillips Model 201) at 12000× magnification.

                                      Table 1     __________________________________________________________________________     Comparison of CooB, CotA and Cf&A Protein Sequences.sup.1     __________________________________________________________________________     CotB  M.KiLlFviL FfnvFaAsAN FMVYPISKDI qsGgSEtIkV FSKSKDVQYI                                             (49)     CooB  MrKLF..LSL LMiPFVAkAN FMIYPISKEI KgGsSELIRI YSKSKDtQYI                                              (48)     CfaA  MhKLFyLLSL LMaPFVAnAN FMIYPISKDl KnGnSELVRV YSKSKEIQYI                                              (50)     Consensus            M-KLF--LSL LM-PFVA-AN FMIYPISKDI K-G-SELIRV YSKSKD-QYI     CotB  KIYTKrVINP GTKEEqEVDI kNWDGGLIVT PaKVVLPAGA SKSIRLTein                                              (99)     CooB  KVYTKKVlNP GTKEEYEVDt PNWEGGLVtT PsKVILPgGg SKSVRLsQLK                                              (98)     CfaA  KIYTKKIINP GTtEEYkVDI PNWDGGLVVT PqKVILPAGA SKSIRLTQFK                                             (100)     Consensus           KIYTKKVINP GTKEEYEVDI PNWDGGLVVT P-KVILPAGA SKSIRLTQ-K     CotB  kkeqEEVYRV YFESVKPggQ DdIeeKNgrv nTDLSVNIIY AALIRtsPen                                             (149)     CooB  dissEDVYRV YFESIKPEkQ Dgl.sKNKsL kTDLSVNIIY AALIRvLPkD                                             (147)     CfaA  ipkkEEVYRV YFEaVKPDsk EnV.idNKKL tTELSVNIIY AALIRsLPsE                                             (149)     Consensus     TDLSVNIIY AALIR-LP-----KNK-L     CotB  pqrkLdVSie sn.NVwIKNT GNIRlGIKDV FLCdtTSI.N DkCAKfsYNr                                             (197)     CooB  gksdMraSls pKssllIKNT GNVRvGIKDa FFCKkTSINn DdCIKKtYNK                                             (197)     CfaA  qnisLnISrn aKkNIiIyNn GNVRaGVKDI YFCKssnI.d DnCVKKaYNK                                             (198)     Consensus     K-N--IKNT GNVR-GIKD- FFCK-TSI-N D-C-KK-YNK     CotB  NlYPDmSvDT kLgkkGFSYa vIDtkDDrnE nsGeLInIKl P                                             (238)     CooB  NIYPgsSFDT gvIqNGFShI FIDsvDgsag KqGkrmlIsI h                                             (238)     CfaA  NIYPEkSFDT .LVnNnFSYV FIklnhEgiE KeqgLIqlKV P                                             (238)     Consensus     L--NGFSY- FID--D---E K-G-LI-IK- P     __________________________________________________________________________      .sup.1 Amino acid sequence comparison was done using the PILEUP algorithm      in the GCG Software. The conserved amino acids are in upper case letters;      nonconserved amino acids are in lower case letters. Amino acids which are      conserved in all three proteins are given in boldface letters in the      consensus amino acid sequence.

                                      TABLE 2     __________________________________________________________________________     Comparison of CooA, CotA and CfaB Protein Sequences.sup.2     __________________________________________________________________________     CotA  MKLnKiIGAL vLsstFVsMG ASAaEKNITV TASVDPTIDL MQSDGtALPS                                              (50)     CooA  MKLKKTIGAM ALaTLFatMG ASAVEKtIsV TASVDPTVDL LQSDGsALPn                                              (50)     CfaB  MKFKKTIGAM ALtTMFVavs ASAVEKNITV TASVDPaIDL LQaDGnALPS                                              (50)     Consensus            MKLKKTIGAM AL-T-FV-MG ASAVEKNITV TASVDPTIDL LQSDG-ALPS     CotA  AVniAYlPge KrFESaRINT QVHTNnkTKG IqIKLtnDnv VMTNlsdPsk                                             (100)     CooA  sVaLtYSPAv nnFEahtINT vVHTNDsdKG VVVKLs.adP VLsNVLNPTl                                              (99)     CfaB  AVkLAYSPAs KtFESyRVmT QVHTNDaTKk VIVKLa.Dtp qLTdVLNsTv                                              (99)     Consensus           AV-LAYSPA- K-FES-RINT QVHTND-TKG V-VKL--D-P VLTNVLNPT-     CotA  tIPleVSFAG tkLSTaAtsI tAdqLNFgAa GVetVSaTkE LVInAgsTq.                                             (149)     CooA  QIPVSVnFAG kpLSTTgitI DsndLNFasS GVNkVSSTQk LsIhAdATrv                                             (149)     CfaB  QmPISVSWgG qvLSTTAkef EAaaLgYsAS GVNgVSSsQE LVIsA.Apkt                                             (148)     Consensus     A--LNF-AS GVN-VSSTQE LVI-A-AT--     CotA  .qTnivAGNY QGLVSIVLTq ep          (170)     CooA  tGgAlTAGqY QGLVSIILTk st          (171)     CfaB  aGTApTAGNY sGvVSlVMTl gs          (170)     Consensus     -TA-TAGNY QGLVSIVLT-     __________________________________________________________________________      .sup.2 Amino acid sequence comparison was done using the PILEUP algorithm      in the GCG Software. The conserved amino acids are in upper case letters;      nonconserved amino acids are in lower case letters. Amino acids which are      conserved in all three proteins are given in boldface letters in the      consensus amino acid sequence.

                                      TABLE 3     __________________________________________________________________________     Comparison of CooC, CotC and CfaC Protein Sequences.sup.3     __________________________________________________________________________     CotC  M. .rafNKIt VfIlfiPGlC fGtnGleskk nIPEeFiDLW mEQDELLEVn                                            (48)     CooC  MiggKssKVV IvlSVliGsS sGfaBkynlv DIPESFRDLW GEQDELLEVr                                            (50)     CfaC  MkhkKkNrlV VaISValipY iGvtG..... DIPDSFRDLW GEQDEFyEVk                                            (45)     Consensus     G--G----- DIPESFRDLW GEQDELLEV-     CotC  LYGrSLGVHR VlTTPTTVKF sSvEeILEKI NVKqEKkeDL RsLLlqSYSR                                            (98)     CooC  LYGQSLGVHR IKsTPTTVaF eSPDnlLDKI eInKgKEaDL RVLMrgSFqR                                            (100)     CfaC  LYGQtLGIHR IKTTPThIKF ySPEsILDKI NlKKEKEkEL sVFFtnSFSR                                            (95)     Consensus     SPE-ILDKI N-KKEKE-DL RVL---SFSR     CotC  NGNMSCnGFd ekeYsCNYIr TdTVnVIVDE enNeLNLFIG asFLsvqAqD                                            (148)     CooC  NGNMSCQGYT g.QnNCNYIK TnTVaVIVDD VENVLNLFIG NEFLaSgenD                                            (149)     CfaC  NGNMSCQGnT tiQYNCNYIK TksVdVIVDD VDNVvNLFIG NEFLdSeAhn                                            (145)     Consensus     -QYNCNYIK T-TV-VIVDD V-NVLNLFIG NEFL-S-A-D     CotC  niYYQkniNs eKAFIHSQTI NFSESegYKs LSlkGvGAqG lTENSYlVFG                                            (198)     CooC  sDYYQpSkNt KKAFIHSQTI NLSDtGnYen LSIvGtGsLG ITDNSYAILG                                            (199)     CfaC  dEYhQlSrNv KKAFIqSQTI NLSDSGkYKr LSIsGnsALG ITDtSYAVLn                                            (195)     Consensus     YYQ-S-N- KKAFIHSQTI NLSDSG-YK- LSI-G-GALG ITDNSYAVLG     CotC  WdAiYNsSrk YtYknQSINn iYYRyDFDKk YYYQLGRMDR SDLSsaSSGN                                            (248)     CooC  WaANYNryks YnYNEQSINS LYFRHDFEKn FYYQLGRiDR SDLSQSSgGN                                            (249)     CfaC  WwmNYNkSng YsnNEktINS LYFRHDLDKr YYYQFGRMDR tDLSQSiSGs                                            (245)     Consensus           W-ANYN-S-- Y-YNEQSINS LYFRHDFDK- YYYQLGRMDR SDLSQSSSGN     CotC  FNFNMLPLPD IDGfqiGTTQ SYIKNiEKSI sSPVTVMLTr FSRVEAFRNE                                            (298)     CooC  FNFdLLPvPD IyGmRaGTTQ SYIKNTgKSV ASPVTIMLTh FSRVEAYRNg                                            (299)     CfaC  FNFNLLPLPD IDGiRtGTTQ SYIKNTDKfI ASPVTVMLTn FSRVEAFRND                                            (295)     Consensus           FNFNLLPLPD IDG-R-GTTQ SYIKNT-KSI ASPVTVMLT- FSRVEAFRN-     CotC  eLLGVWYLnS GINDLDTsRL PDGSYDLtLK IFEQDiLVRE EkVPFNKGGa                                            (348)     CooC  QLLGVWYLDa GIsELDTeRL PDGnYDLKLK IFEQEQLVRE EIVPFNKsGS                                            (349)     CfaC  QLLGVWYLDS GVNELDTaRL PyGSYDLKLK IFEntQLVRE EIIPFNKGrS                                            (345)     Consensus           QLLGVWYLDS GINELDT-RL PDGSYDLKLK IFEQ-QLVRE EIVPFNKGGS     CotC  SfGDMQWDVF aQAGNIVNnN DsYIEKQtNk KtgINaGiRt PVTRNLSflQ                                            (398)     COOC  SIGDthWDVF VQAGdIINDN gRYVEKQkNH KSaINsGLRL PlTRNLaVQl                                            (399)     CfaC  SIGDMQWDIF VQgGNIVNDN DRYIEKQnNH KSsINtGLRL PITkNiSVQQ                                            (395)     Consensus           SIGDMQWDVF VQAGNIVNDN DRYIEKQ-NH KS-IN-GLRL P-TRNLSVQQ     CotC  GGAIIDNdkY YEaGVnWrSG FLDGvLsgnF SFLYGDgArG NYQNISYTDG                                            (448)     CooC  GGAVIDNKnY YEtGIlWNSG LLDGSLNSkF tFLFGDdthG NYQNVSYTDG                                            (449)     CfaC  GvsVIDNKsY YEgslkWNSG iLsGSLNSeF SFLWGDnAkG NYQsISYTDG                                            (445)     Consensus     LDGSLNS-F SFL-GD-A-G NYQNISYTDG     CotC  FnLSFYrNDK sVDNCshNYs AGWSGCYESY SfSLSVPVsG WTtTLGYnhT                                            (498)     CooC  FSLSFYHNDK RVDdCGkdYN mGWSGCYESY SASLSIPVkG WnSTLaYSnT                                            (499)     CfaC  FSLSFYHNDK RVDNCGrNYN AGWSGCYESY SASLSIPllG WTSTLGYSdT                                            (495)     Consensus           FSLSFYHNDK RVDNCG-NYN AGWSGCYESY SASLSIPV-G WTSTLGYS-T     CotC  nnEaVhKYDy tpEY..Ffsk kYKGvsKRWQ LTSSsSyKWM DYhViPTIGV                                            (546)     CooC  YStSVYrYDa vSEYvpY..y yYKGRTKRWQ LTaSTvvrWg DYNIMPTIGV                                            (547)     CfaC  YSESVYKshi lSEYgfYnqn iYKGRTqRWQ LTSSTSlKWM DYNfMPaIGI                                            (545)     Consensus     YKGRTKRWQ LTSSTS-KWM DYN-MPTIGV     CotC  YrSDQsrWsE qGGYFSLsFT RVkensaiNA GYSYNYvkhk nathEAFlDG                                            (596)     CooC  YNSEQkQWaD KGGYLSLTLT RVdggkSLNA GYSYNYSRGN YtSNDAFVEG                                            (597)     CfaC  YNSRQrQltD KGGYiSvTiT RasrenSLNt GYSYNYSRGN YsSNElFVDG                                            (595)     Consensus           YNSEQ-QW-D KGGY-SLT-T RV----SLNA GYSYNYSRGN Y-SNEAFVDG     CotC  riT.TNtfgY sELGsRINtN knNTEaGVtG RVkNRFGDLN GSLNVNKSkt                                            (645)     CooC  hLvSdtNvSY rELsaRVsgN RyyTEGGVSG RINNRFGDLN GtLNVNKNRk                                            (647)     CfaC  yMTSTNNgdY hEaGmRfNkN RhNaEGrlSG RINNRFGDLN GSFsmNKNRn                                            (645)     Consensus     ELG-R-N-N R-NTEGGVSG RINNRFGDLN GSLNVNKNR-     CotC  SgkmTHSMsA nYNSSFAiTg DsVYWGGdAS GLTKLSGGVV nVrSdDksKE                                            (695)     CooC  ShdTTHSLTA GYsSSFALTt DGIYWGGSAS GLTnLSGGIV rVKSNEdesE                                            (697)     CfaC  tnsTnHSLTg GYNSSFALTs DGfYWGGSta GLTKLaGGII kVKSNDtkKn                                            (695)     Consensus     VKSND--KE-TTHSLTA GYNSSFALT- DG YWGGSAS GLTKLSGGIV     CotC  LIKIsGSSYG nYiLGSNDrs FIPVsALMPs nLTIEEiqsn DKNItVqAls                                            (745)     CooC  LinVkGSSYG hYSLGSNDsl FIPVPALMqA SLTIEENtnk sKNIdVLAPT                                            (747)     CfaC  LVKVtGtlYG dYSLGSNDna FIPVPALtPA SLiIEDNnyg DnNIsILAPT                                            (745)     Consensus     YSLGSND--FIPVPALMPA SLTIEEN--- DKNI-VLAPT     CotC  KNDFFiLPGN VFPIDVtANV tVSYIGRaLD dKGnPLSnAH ILdvhgVrLD                                            (795)     CooC  KNtFFMLPGs VYPIDVsANV SftYVGRGvD vKGrPLSGAy ILNaqnIVLD                                            (797)     CfaC  nNDmFMLPGN VYPVEIetkV SVSYIGRGFD pnGtPLSGAH VLNephVILD                                            (795)     Consensus     KG-PLSGAH ILN---V-LDIDV-ANV SVSYIGRG-D     CotC  EDGGFSFEtS aqkKsLFLLK DKdIYSCdvk KyDLRsGVlF tGDlICEhsg                                            (845)     CooC  EnGGFSFEsS eNEKeLFLLK DKTIYSCsLd rsEMRnGIaF VGEVaCnsti                                            (847)     CfaC  EDGGFSFEyt gNEKtLFLLK grTIYtCqLg KnkvhkGlvF VGDVICDins                                            (845)     Consensus     NEK-LFLLK DKTIYSC-L- K---R-GI-F VGDVIC ----     CotC  iErLgkDLVn NPRVkqLLAY k....      (866)     CooC  kElLPEkLVt NsRIhDLLAY Nqdte      (872)     CfaC  tssLPDEFVk NPRVqDLLAk Ndkg.      (869)     Consensus     E-LP--LV- NPRV-DLLAY N----     __________________________________________________________________________      .sup.3 Amino acid sequence comparison was done using the PILEUP algorithm      in the GCG Software. The conserved amino acids are in upper Case letters;      nonconserved amino acids are in lower case 1etters. Amino acids which are      Conserved in all three proteins are given in boldface letters in the      consensus amino acid sequence

                                      TABLE 4     __________________________________________________________________________     Comparison of CooD, CotD and CfaE Protein Sequences.sup.4     __________________________________________________________________________     CotD  LKKViFVLSM FLcSq..vYg qSwhtNvEag siNkTeSIGP idrsaaaSyP                                            (48)     CooD  MKKIFifLSi iFSa.....v VSAgryPEtt vgNLTkSfqa prlDRsvqSP                                            (45)     CfaE  MnKILFIFtL FFSSgfftFa VSAdkNPgs. .eNMTntIGP h..DRggSSP                                            (46)     Consensus     --DR--SSP I-F-LS- FFSS------ VSA--NPE--     CotD  ahyIFheHVA GYNkdHSLFD RMtFLCMSSt daskGACPTg EnSkss..qG                                            (96)     CooD  IYNIFtnHVA GYslSHSLYD RivFLCtSSs NpNGACPTi gtSGvqy..G                                            (93)     CfaE  IYNILnsylt aYNgSHhLYD RMsFLCLSSq NtlNGACPss DapGtatidG                                            (96)     Consensus     -SG-----GIF--HVA GYN-SHSLYD RM-FLC-SS- N--NGACPT-     CotD  ETNIkLiFTE KkSLarktLN LKGYKrFLYE sdrCihYvdK MnLNShtvkC                                            (146)     CooD  tTtITLQFTE KRSLIKRniN LaGnKkpiWE NqsC.dFsnl MvLNSksWsC                                            (142)     CfaE  ETNITLQFTE KRSLIKReLq iKGYKqFLFk NanC...psK LaLNSshFqC                                            (143)     Consensus           ETNITLQFTE KRSLIKR-LN LKGYK-FL-E N--C-----K M-LNS----C     CotD  vg.sftrGvd FtLYIPqGEI dgLltGGIWE ATLeLRVKRh YDy...nhGT                                            (192)     CooD  gahgnAnGtl LnLYIPAGEI NKLPFGGIWE ATLiLRlsRy gEvssThYGn                                            (192)     CfaE  n.reqAsGat LsLYIPAGEl NKLPFGGVWn AvLkLnVKRr YD...TtYGT                                            (189)     Consensus     A-G-- L-LYIPAGEI NKLPFGGIWE ATL-LRVKR- YD---T-YGT     CotD  YkVNITVDLT DKGNIQVWtP kFHSdPRIDL NLRPeGNGKY SGSNvLEMCL                                            (242)     CooD  YTVNITVDLT DKGNIQVWLP gFHSNPRVDL NLRPiGNyKY SGSNSLDMCF                                            (242)     CfaE  YTINITVnLT DKGNIQIWLP qFkSNaRVDL NLRPtGgGtY iGrNSvDMCF                                            (239)     Consensus     FHSNPRVDL NLRP-GNGKY SGSNSLDMCF     CotD  YDGYSThSqS iEmRFQDDSq TgnnEYNLiK tGEplKkLPY kLSLLLGGre                                            (292)     CooD  YDGYSTNSdS MvIkFQDDNp TnssEYNLyK IGgteK.LPY avSLLMGeKi                                            (291)     CfaE  YDGYSTNSsS LEIRFQDDNs ksdgkFyLkK InDdsKeLvY tLSLLLaGKn                                            (289)     Consensus     LSLLLGGK- T---EYNL-K IG---K-LPY     CotD  FYPnNGeAFT INDtSsLfiN WNRIksVsLP qIsIPVLCWP AnLtFmsElN                                            (342)     CooD  FYPvNGQsFT INDsSvLETN WNRVTAVaMP EVnVPVLCWP ARLlLnADVN                                            (341)     CfaE  LtPtNGQALn INtaS.LETN WNRITAVtMP EISVPVLCWP gRLqLdakVk                                            (338)     Consensus           FYP-NGQAFT IND-S-LETN WNRITAV-MP EISVPVLCMP ARL-L-A-VN     CotD  aPDAGQYSGq IyITFTPSSS SL         (364)     CooD  NPEAGQYmGn IkITFTPSSq tL         (363)     CfaE  NPEAGeYSGi lnVTFTPSSS SL         (36O)     Consensus           NPEAGQYSG- I-ITFTPSSS SL     __________________________________________________________________________      .sup.4 Amino acid sequence comparison was done using the PILEUP algorithm      in the GCG SoftWare. The conserved amino acids are in upper case letters;      nonconserved amino acids are in lower case letters. Amino acids which are      conserved in all three proteins are given in boldface letters in the      consensus amino acid sequence.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 17     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 5798 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: double               (D) TOPOLOGY: circular     -     (ii) MOLECULE TYPE: DNA (genomic)     -    (iii) HYPOTHETICAL: NO     -     (iv) ANTI-SENSE: NO     -     (vi) ORIGINAL SOURCE:     #coli     (A) ORGANISM: Escherichia     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 499..1215     -     (ix) FEATURE:               (A) NAME/KEY: sig.sub.-- - #peptide               (B) LOCATION: 499..552     -     (ix) FEATURE:               (A) NAME/KEY: mat.sub.-- - #peptide               (B) LOCATION: 553..1212     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 1255..1767     -     (ix) FEATURE:               (A) NAME/KEY: sig.sub.-- - #peptide               (B) LOCATION: 1255..1323     -     (ix) FEATURE:               (A) NAME/KEY: mat.sub.-- - #peptide               (B) LOCATION: 1324..1764     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 1836..4436     -     (ix) FEATURE:               (A) NAME/KEY: sig.sub.-- - #peptide               (B) LOCATION: 1836..1913     -     (ix) FEATURE:               (A) NAME/KEY: mat.sub.-- - #peptide               (B) LOCATION: 1914..4433     -     (ix) FEATURE:               (A) NAME/KEY: CDS               (B) LOCATION: 4451..5545     -     (ix) FEATURE:               (A) NAME/KEY: sig.sub.-- - #peptide               (B) LOCATION: 4451..4504     -     (ix) FEATURE:               (A) NAME/KEY: mat.sub.-- - #peptide               (B) LOCATION: 4505..5542     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     - CTGCAGATGG CGCTGTGGCG GCGTAAGAGG CCCCGGAACG TTATCGTTCA CA - #CGGACCGT       60     - GGAGGCCAGT ACTGTTCAGC AGATTATCAG GCGCAACTGA AGCGGCATAA TC - #TGCGTGGA      120     - AGTATGAGCG CAAAAGGTTG CTGCTACGAT AATGCCTGCG TGGAAAGCTT CT - #TTCATTCG      180     - CTGAAAGTGG AATGTATCCA TGGAGAACAC TTTATGAGCC GGGAAATAAG TC - #GGGCAACG      240     - GTGTTTAATT ATATCGAATG TGATTACAAT CGGTGGCGGC GGCACAGTTG GT - #GTGGCGGC      300     - CTCAGTCCGG AACAATTTGA AAACAAGAAC CTCGCTTAGG CCTGTGTCCA TA - #TTACGTGG      360     - GTAGGATCAA AACACTATCA ATAAGTTGGA GTCATTACCG GCATTCTTGA AA - #GCCTCATG      420     - CGTGACAGGG TGTGTGTTGT ATTTTTATCA TATTTTAACG CCTGCTTTCT GA - #TAATGTTT      480     #ATT CTG TTT TTT      531 AAG ATA TTG TTA TTT GTT     #Phe Pheys Ile Leu Leu Phe Val Ile Leu     10     - AAT GTT TTT GCT GCC AGT GCA AAT TTT ATG GT - #A TAT CCG ATC TCA AAG      579     Asn Val Phe Ala Ala Ser Ala Asn Phe Met Va - #l Tyr Pro Ile Ser Lys     #       5  1     - GAT ATA CAG AGT GGT GGC AGC GAA ACT ATA AA - #A GTT TTT TCA AAA TCT      627     Asp Ile Gln Ser Gly Gly Ser Glu Thr Ile Ly - #s Val Phe Ser Lys Ser     # 25     - AAA GAT GTT CAG TAT ATA AAG ATA TAT ACG AA - #A AGG GTT ATT AAT CCA      675     Lys Asp Val Gln Tyr Ile Lys Ile Tyr Thr Ly - #s Arg Val Ile Asn Pro     #                 40     - GGA ACA AAA GAA GAG CAA GAG GTT GAT ATA AA - #A AAT TGG GAT GGA GGT      723     Gly Thr Lys Glu Glu Gln Glu Val Asp Ile Ly - #s Asn Trp Asp Gly Gly     #             55     - CTG ATT GTA ACT CCG GCA AAA GTT GTT TTG CC - #A GCT GGA GCA AGT AAG      771     Leu Ile Val Thr Pro Ala Lys Val Val Leu Pr - #o Ala Gly Ala Ser Lys     #         70     - TCA ATA CGA CTT ACT GAG ATA AAT AAA AAA GA - #G CAG GAG GAA GTC TAT      819     Ser Ile Arg Leu Thr Glu Ile Asn Lys Lys Gl - #u Gln Glu Glu Val Tyr     #     85     - CGT GTG TAT TTT GAA TCT GTA AAA CCG GGA CA - #G CAA GAT GAT ATA GAG      867     Arg Val Tyr Phe Glu Ser Val Lys Pro Gly Gl - #n Gln Asp Asp Ile Glu     #105     - GAA AAA AAT GGG CGT GTA AAT ACT GAT TTA TC - #A GTA AAC ATA ATC TAT      915     Glu Lys Asn Gly Arg Val Asn Thr Asp Leu Se - #r Val Asn Ile Ile Tyr     #               120     - GCC GCT CTC ATA AGA ACC AGC CCT GAG AAC CC - #A CAG AGG AAA CTT GAT      963     Ala Ala Leu Ile Arg Thr Ser Pro Glu Asn Pr - #o Gln Arg Lys Leu Asp     #           135     - GTA TCC ATA GAA TCA AAC AAT GTA TGG ATT AA - #G AAC ACT GGA AAT ATT     1011     Val Ser Ile Glu Ser Asn Asn Val Trp Ile Ly - #s Asn Thr Gly Asn Ile     #       150     - AGG CTG GGA ATT AAG GAT GTA TTC TTG TGT GA - #T ACA ACC AGC ATA AAT     1059     Arg Leu Gly Ile Lys Asp Val Phe Leu Cys As - #p Thr Thr Ser Ile Asn     #   165     - GAT AAA TGT GCA AAG TTT TCT TAT AAT AGA AA - #T CTA TAT CCA GAT ATG     1107     Asp Lys Cys Ala Lys Phe Ser Tyr Asn Arg As - #n Leu Tyr Pro Asp Met     170                 1 - #75                 1 - #80                 1 -     #85     - TCG GTA GAT ACT AAA TTA GGA AAA AAA GGA TT - #T TCT TAT GCT GTC ATT     1155     Ser Val Asp Thr Lys Leu Gly Lys Lys Gly Ph - #e Ser Tyr Ala Val Ile     #               200     - GAT ACA AAG GAT GAC AGA AAT GAA AAT AGC GG - #A GAG TTA ATT AAC ATA     1203     Asp Thr Lys Asp Asp Arg Asn Glu Asn Ser Gl - #y Glu Leu Ile Asn Ile     #           215     - AAG CTC CCG TAA GATAAATTGT TCAATAACCA CTGTATAAGG GT - #GTAAATA ATG     1257     #               Met     23     - AAA CTC AAT AAG ATT ATT GGA GCA TTA GTT CT - #T TCA TCT ACA TTT GTT     1305     Lys Leu Asn Lys Ile Ile Gly Ala Leu Val Le - #u Ser Ser Thr Phe Val     10     - AGC ATG GGG GCT TCT GCT GCC GAG AAA AAT AT - #C ACT GTA ACT GCT AGC     1353     Ser Met Gly Ala Ser Ala Ala Glu Lys Asn Il - #e Thr Val Thr Ala Ser     #   10     - GTT GAT CCA ACT ATC GAT CTG ATG CAA TCT GA - #T GGC ACA GCG TTA CCA     1401     Val Asp Pro Thr Ile Asp Leu Met Gln Ser As - #p Gly Thr Ala Leu Pro     #                 25     - AGT GCA GTT AAT ATT GCA TAT CTT CCA GGA GA - #G AAA AGA TTT GAA TCT     1449     Ser Ala Val Asn Ile Ala Tyr Leu Pro Gly Gl - #u Lys Arg Phe Glu Ser     #             40     - GCT CGT ATC AAT ACC CAA GTT CAT ACC AAT AA - #T AAA ACT AAG GGT ATT     1497     Ala Arg Ile Asn Thr Gln Val His Thr Asn As - #n Lys Thr Lys Gly Ile     #         55     - CAG ATA AAG CTT ACT AAT GAT AAT GTG GTA AT - #G ACT AAC TTA TCT GAT     1545     Gln Ile Lys Leu Thr Asn Asp Asn Val Val Me - #t Thr Asn Leu Ser Asp     #     70     - CCA AGC AAG ACT ATT CCT TTA GAG GTT TCA TT - #C GCT GGC ACT AAG CTG     1593     Pro Ser Lys Thr Ile Pro Leu Glu Val Ser Ph - #e Ala Gly Thr Lys Leu     # 90     - AGC ACA GCT GCA ACA TCT ATT ACT GCC GAT CA - #A TTA AAT TTT GGC GCA     1641     Ser Thr Ala Ala Thr Ser Ile Thr Ala Asp Gl - #n Leu Asn Phe Gly Ala     #                105     - GCT GGT GTA GAG ACA GTT TCT GCA ACT AAG GA - #A CTC GTT ATT AAT GCA     1689     Ala Gly Val Glu Thr Val Ser Ala Thr Lys Gl - #u Leu Val Ile Asn Ala     #           120     - GGA AGC ACC CAG CAA ACT AAT ATT GTA GCT GG - #T AAC TAT CAA GGA TTG     1737     Gly Ser Thr Gln Gln Thr Asn Ile Val Ala Gl - #y Asn Tyr Gln Gly Leu     #       135     - GTG TCA ATT GTG CTT ACT CAA GAA CCT TAA TA - #AACATTAA GATATATCAA     1787     Val Ser Ile Val Leu Thr Gln Glu Pro  *     #   145     #CGA GCT    1844TTTTTAG TCACCCTGTT ATTAAAGAAA ATATATTT ATG     #                 Met - # Arg Ala     25 - #     - TTC AAT AAA ATA ACT GTT TTC ATT TTG TTT AT - #T CCT GGT TTA TGT TTT     1892     Phe Asn Lys Ile Thr Val Phe Ile Leu Phe Il - #e Pro Gly Leu Cys Phe     10     - GGA ACG AAT GGT TTA GAG AGT AAA AAA AAT AT - #T CCT GAA GAA TTT ATA     1940     Gly Thr Asn Gly Leu Glu Ser Lys Lys Asn Il - #e Pro Glu Glu Phe Ile     #       5  1     - GAC TTA TGG ATG GAA CAG GAT GAA TTA CTT GA - #A GTT AAT TTA TAT GGG     1988     Asp Leu Trp Met Glu Gln Asp Glu Leu Leu Gl - #u Val Asn Leu Tyr Gly     # 25     - CGT TCT CTA GGT GTT CAT CGT GTA TTG ACA AC - #G CCT ACT ACT GTG AAA     2036     Arg Ser Leu Gly Val His Arg Val Leu Thr Th - #r Pro Thr Thr Val Lys     #                 40     - TTT TCA TCT GTA GAG GAA ATT CTA GAA AAG AT - #T AAT GTG AAA CAA GAG     2084     Phe Ser Ser Val Glu Glu Ile Leu Glu Lys Il - #e Asn Val Lys Gln Glu     #             55     - AAA AAA GAA GAC CTG AGA AGT CTT CTT CTT CA - #A TCA TAT TCC CGC AAC     2132     Lys Lys Glu Asp Leu Arg Ser Leu Leu Leu Gl - #n Ser Tyr Ser Arg Asn     #         70     - GGG AAT ATG AGT TGT AAT GGG TTT GAT GAA AA - #G GAA TAT AGC TGC AAT     2180     Gly Asn Met Ser Cys Asn Gly Phe Asp Glu Ly - #s Glu Tyr Ser Cys Asn     #     85     - TAC ATT AGA ACT GAT ACG GTT AAT GTT ATT GT - #A GAT GAA GAA AAT AAT     2228     Tyr Ile Arg Thr Asp Thr Val Asn Val Ile Va - #l Asp Glu Glu Asn Asn     #105     - GAG CTA AAT CTT TTT ATA GGT GCG AGT TTT CT - #T TCT GTT CAA GCT CAG     2276     Glu Leu Asn Leu Phe Ile Gly Ala Ser Phe Le - #u Ser Val Gln Ala Gln     #               120     - GAT AAT ATT TAT TAT CAA AAA AAT ATA AAC TC - #A GAA AAA GCA TTC ATT     2324     Asp Asn Ile Tyr Tyr Gln Lys Asn Ile Asn Se - #r Glu Lys Ala Phe Ile     #           135     - CAC AGT CAG ACA ATT AAC TTT TCT GAA TCT GA - #A GGG TAT AAA AGT TTA     2372     His Ser Gln Thr Ile Asn Phe Ser Glu Ser Gl - #u Gly Tyr Lys Ser Leu     #       150     - TCT TTG AAA GGG GTT GGT GCA CAG GGG TTA AC - #T GAA AAT AGT TAT CTT     2420     Ser Leu Lys Gly Val Gly Ala Gln Gly Leu Th - #r Glu Asn Ser Tyr Leu     #   165     - GTT TTT GGT TGG GAT GCC ATA TAT AAT AGT TC - #T AGG AAA TAC ACA TAT     2468     Val Phe Gly Trp Asp Ala Ile Tyr Asn Ser Se - #r Arg Lys Tyr Thr Tyr     170                 1 - #75                 1 - #80                 1 -     #85     - AAA AAT CAG TCA ATC AAT AAT ATA TAT TAC AG - #A TAT GAT TTT GAT AAA     2516     Lys Asn Gln Ser Ile Asn Asn Ile Tyr Tyr Ar - #g Tyr Asp Phe Asp Lys     #               200     - AAA TAT TAT TAT CAG TTG GGG CGA ATG GAT CG - #T TCA GAT TTA TCA AGT     2564     Lys Tyr Tyr Tyr Gln Leu Gly Arg Met Asp Ar - #g Ser Asp Leu Ser Ser     #           215     - GCC TCT AGT GGT AAT TTT AAT TTC AAT ATG CT - #T CCT TTG CCT GAT ATT     2612     Ala Ser Ser Gly Asn Phe Asn Phe Asn Met Le - #u Pro Leu Pro Asp Ile     #       230     - GAT GGA TTT CAG ATA GGT ACG ACC CAA TCC TA - #T ATT AAA AAT ATC GAA     2660     Asp Gly Phe Gln Ile Gly Thr Thr Gln Ser Ty - #r Ile Lys Asn Ile Glu     #   245     - AAA TCA ATA TCA TCG CCA GTA ACC GTT ATG TT - #A ACC CGA TTT TCT AGG     2708     Lys Ser Ile Ser Ser Pro Val Thr Val Met Le - #u Thr Arg Phe Ser Arg     250                 2 - #55                 2 - #60                 2 -     #65     - GTT GAA GCC TTT CGT AAT GAA GAG TTA CTG GG - #A GTA TGG TAT TTG AAT     2756     Val Glu Ala Phe Arg Asn Glu Glu Leu Leu Gl - #y Val Trp Tyr Leu Asn     #               280     - TCA GGA ATC AAT GAT CTC GAT ACA AGT CGT TT - #G CCT GAC GGC AGT TAT     2804     Ser Gly Ile Asn Asp Leu Asp Thr Ser Arg Le - #u Pro Asp Gly Ser Tyr     #           295     - GAT TTA ACG TTG AAG ATA TTT GAG CAG GAC AT - #T CTT GTT CGT GAA GAG     2852     Asp Leu Thr Leu Lys Ile Phe Glu Gln Asp Il - #e Leu Val Arg Glu Glu     #       310     - AAG GTC CCT TTT AAC AAG GGA GGA GCC TCT TT - #T GGG GAT ATG CAA TGG     2900     Lys Val Pro Phe Asn Lys Gly Gly Ala Ser Ph - #e Gly Asp Met Gln Trp     #   325     - GAT GTG TTT GCT CAG GCT GGT AAT ATT GTC AA - #T AAT AAC GAT AGT TAT     2948     Asp Val Phe Ala Gln Ala Gly Asn Ile Val As - #n Asn Asn Asp Ser Tyr     330                 3 - #35                 3 - #40                 3 -     #45     - ATT GAG AAG CAA ACT AAT AAA AAA ACG GGA AT - #A AAT GCT GGT ATA CGT     2996     Ile Glu Lys Gln Thr Asn Lys Lys Thr Gly Il - #e Asn Ala Gly Ile Arg     #               360     - ACG CCT GTA ACC AGA AAT TTA TCG TTC TTA CA - #G GGC GGT GCT ATA ATT     3044     Thr Pro Val Thr Arg Asn Leu Ser Phe Leu Gl - #n Gly Gly Ala Ile Ile     #           375     - GAT AAT GAT AAA TAT TAT GAG GCT GGT GTT AA - #C TGG CGT TCA GGG TTT     3092     Asp Asn Asp Lys Tyr Tyr Glu Ala Gly Val As - #n Trp Arg Ser Gly Phe     #       390     - CTT GAT GGG GTA CTA AGT GGA AAC TTC AGT TT - #C CTG TAT GGT GAT GGT     3140     Leu Asp Gly Val Leu Ser Gly Asn Phe Ser Ph - #e Leu Tyr Gly Asp Gly     #   405     - GCA AGA GGA AAT TAT CAA AAT ATT TCG TAT AC - #C GAT GGT TTT AAT CTC     3188     Ala Arg Gly Asn Tyr Gln Asn Ile Ser Tyr Th - #r Asp Gly Phe Asn Leu     410                 4 - #15                 4 - #20                 4 -     #25     - TCT TTT TAT CGT AAT GAT AAA AGC GTT GAT AA - #T TGT AGT CAC AAT TAC     3236     Ser Phe Tyr Arg Asn Asp Lys Ser Val Asp As - #n Cys Ser His Asn Tyr     #               440     - AGT GCG GGA TGG AGT GGG TGC TAT GAG TCT TA - #T TCC TTT TCA CTA AGT     3284     Ser Ala Gly Trp Ser Gly Cys Tyr Glu Ser Ty - #r Ser Phe Ser Leu Ser     #           455     - GTT CCT GTA TCT GGC TGG ACT ACT ACT CTT GG - #C TAT AAC CAT ACA AAT     3332     Val Pro Val Ser Gly Trp Thr Thr Thr Leu Gl - #y Tyr Asn His Thr Asn     #       470     - AAT GAG GCT GTA CAT AAA TAT GAT TAC ACC CC - #G GAA TAT TTT TTT AGT     3380     Asn Glu Ala Val His Lys Tyr Asp Tyr Thr Pr - #o Glu Tyr Phe Phe Ser     #   485     - AAA AAA TAT AAA GGT GTC AGT AAA AGA TGG CA - #A TTG ACA TCT TCT TCG     3428     Lys Lys Tyr Lys Gly Val Ser Lys Arg Trp Gl - #n Leu Thr Ser Ser Ser     490                 4 - #95                 5 - #00                 5 -     #05     - TCC TAT AAA TGG ATG GAT TAT CAT GTG ATT CC - #G ACG ATA GGT GTA TAT     3476     Ser Tyr Lys Trp Met Asp Tyr His Val Ile Pr - #o Thr Ile Gly Val Tyr     #               520     - CGT AGT GAT CAG AGT CGA TGG AGT GAG CAG GG - #A GGG TAT TTT TCT TTG     3524     Arg Ser Asp Gln Ser Arg Trp Ser Glu Gln Gl - #y Gly Tyr Phe Ser Leu     #           535     - AGT TTT ACC CGA GTA AAG GAA AAT AGT GCC AT - #T AAT GCA GGA TAT TCT     3572     Ser Phe Thr Arg Val Lys Glu Asn Ser Ala Il - #e Asn Ala Gly Tyr Ser     #       550     - TAT AAT TAT GTA AAG CAT AAA AAT GCC ACA CA - #T GAG GCT TTT TTA GAT     3620     Tyr Asn Tyr Val Lys His Lys Asn Ala Thr Hi - #s Glu Ala Phe Leu Asp     #   565     - GGT CGT ATA ACG ACA AAT ACT TTT GGC TAT AG - #T GAA TTA GGC TCT CGT     3668     Gly Arg Ile Thr Thr Asn Thr Phe Gly Tyr Se - #r Glu Leu Gly Ser Arg     570                 5 - #75                 5 - #80                 5 -     #85     - ATA AAT ACG AAC AAA AAT AAC ACA GAA GCA GG - #T GTT ACC GGA CGT GTA     3716     Ile Asn Thr Asn Lys Asn Asn Thr Glu Ala Gl - #y Val Thr Gly Arg Val     #               600     - AAA AAC AGG TTT GGA GAT CTG AAT GGT TCA TT - #A AAT GTT AAT AAA AGT     3764     Lys Asn Arg Phe Gly Asp Leu Asn Gly Ser Le - #u Asn Val Asn Lys Ser     #           615     - AAA ACA TCC GGT AAG ATG ACT CAC TCA ATG AG - #T GCA AAC TAT AAC TCC     3812     Lys Thr Ser Gly Lys Met Thr His Ser Met Se - #r Ala Asn Tyr Asn Ser     #       630     - TCA TTT GCA ATT ACT GGT GAT TCT GTC TAT TG - #G GGG GGA GAT GCC TCT     3860     Ser Phe Ala Ile Thr Gly Asp Ser Val Tyr Tr - #p Gly Gly Asp Ala Ser     #   645     - GGT TTA ACG AAG CTA TCT GGG GGT GTG GTG AA - #T GTA AGA TCA GAT GAT     3908     Gly Leu Thr Lys Leu Ser Gly Gly Val Val As - #n Val Arg Ser Asp Asp     650                 6 - #55                 6 - #60                 6 -     #65     - AAA TCA AAA GAG CTA ATA AAA ATA TCA GGT TC - #T TCA TAT GGT AAT TAT     3956     Lys Ser Lys Glu Leu Ile Lys Ile Ser Gly Se - #r Ser Tyr Gly Asn Tyr     #               680     - ATC CTC GGC AGT AAT GAC CGT TCA TTT ATC CC - #T GTA AGT GCA TTA ATG     4004     Ile Leu Gly Ser Asn Asp Arg Ser Phe Ile Pr - #o Val Ser Ala Leu Met     #           695     - CCA AGT AAC CTA ACT ATA GAA GAG ATT CAG TC - #A AAC GAC AAG AAT ATT     4052     Pro Ser Asn Leu Thr Ile Glu Glu Ile Gln Se - #r Asn Asp Lys Asn Ile     #       710     - ACT GTT CAG GCG TTA TCA AAA AAT GAC TTT TT - #T ATT CTG CCT GGT AAT     4100     Thr Val Gln Ala Leu Ser Lys Asn Asp Phe Ph - #e Ile Leu Pro Gly Asn     #   725     - GTT TTC CCT ATT GAT GTA ACT GCT AAT GTG AC - #A GTT TCT TAT ATA GGG     4148     Val Phe Pro Ile Asp Val Thr Ala Asn Val Th - #r Val Ser Tyr Ile Gly     730                 7 - #35                 7 - #40                 7 -     #45     - AGA GCT CTT GAT GAT AAA GGA AAT CCA TTA TC - #A AAT GCC CAT ATA CTT     4196     Arg Ala Leu Asp Asp Lys Gly Asn Pro Leu Se - #r Asn Ala His Ile Leu     #               760     - GAT GTT CAC GGG GTT AGG CTG GAT GAG GAT GG - #T GGT TTT TCT TTC GAA     4244     Asp Val His Gly Val Arg Leu Asp Glu Asp Gl - #y Gly Phe Ser Phe Glu     #           775     - ACT TCA GCT CAA AAG AAA TCT CTT TTC CTG TT - #A AAA GAT AAA GAT ATT     4292     Thr Ser Ala Gln Lys Lys Ser Leu Phe Leu Le - #u Lys Asp Lys Asp Ile     #       790     - TAT TCA TGT GAT GTT AAG AAA TAT GAT TTA CG - #T AGT GGT GTT TTA TTT     4340     Tyr Ser Cys Asp Val Lys Lys Tyr Asp Leu Ar - #g Ser Gly Val Leu Phe     #   805     - ACT GGT GAC CTT ATA TGT GAA CAC AGT GGT AT - #A GAA CGT CTT GGA AAA     4388     Thr Gly Asp Leu Ile Cys Glu His Ser Gly Il - #e Glu Arg Leu Gly Lys     810                 8 - #15                 8 - #20                 8 -     #25     - GAT TTG GTT AAC AAT CCA AGA GTT AAG CAA CT - #G CTT GCT TAT AAA TAA     4436     Asp Leu Val Asn Asn Pro Arg Val Lys Gln Le - #u Leu Ala Tyr Lys  *     #               840     - CCAAGAGGTG AACT TTG AAA AAA GTG ATT TTT GTT T - #TA TCC ATG TTT CTA     4486                     Leu L - #ys Lys Val Ile Phe Val Leu Ser Met Phe Le - #u     10     - TGT TCT CAG GTT TAC GGG CAA TCA TGG CAT AC - #G AAC GTA GAG GCT GGT     4534     Cys Ser Gln Val Tyr Gly Gln Ser Trp His Th - #r Asn Val Glu Ala Gly     #   10     - TCA ATA AAT AAA ACA GAG TCG ATA GGC CCC AT - #A GAC CGA AGT GCT GCT     4582     Ser Ile Asn Lys Thr Glu Ser Ile Gly Pro Il - #e Asp Arg Ser Ala Ala     #                 25     - GCA TCG TAT CCT GCT CAT TAT ATA TTT CAT GA - #A CAT GTT GCT GGT TAC     4630     Ala Ser Tyr Pro Ala His Tyr Ile Phe His Gl - #u His Val Ala Gly Tyr     #             40     - AAT AAA GAT CAC TCT CTT TTT GAC AGG ATG AC - #G TTT TTA TGT ATG TCA     4678     Asn Lys Asp His Ser Leu Phe Asp Arg Met Th - #r Phe Leu Cys Met Ser     #         55     - TCA ACA GAT GCA TCT AAA GGT GCA TGT CCG AC - #A GGA GAA AAC TCC AAA     4726     Ser Thr Asp Ala Ser Lys Gly Ala Cys Pro Th - #r Gly Glu Asn Ser Lys     #     70     - TCC TCT CAA GGG GAG ACT AAT ATT AAG CTA AT - #A TTT ACT GAA AAG AAA     4774     Ser Ser Gln Gly Glu Thr Asn Ile Lys Leu Il - #e Phe Thr Glu Lys Lys     # 90     - AGT CTG GCC AGA AAA ACA TTA AAC TTA AAA GG - #A TAT AAG AGA TTT TTA     4822     Ser Leu Ala Arg Lys Thr Leu Asn Leu Lys Gl - #y Tyr Lys Arg Phe Leu     #                105     - TAT GAA TCA GAT AGA TGC ATT CAT TAT GTC GA - #T AAA ATG AAT CTC AAT     4870     Tyr Glu Ser Asp Arg Cys Ile His Tyr Val As - #p Lys Met Asn Leu Asn     #           120     - TCT CAT ACT GTT AAA TGT GTA GGT TCA TTC AC - #A AGA GGA GTA GAT TTC     4918     Ser His Thr Val Lys Cys Val Gly Ser Phe Th - #r Arg Gly Val Asp Phe     #       135     - ACT TTA TAT ATC CCA CAA GGT GAA ATT GAT GG - #G CTT CTA ACT GGA GGT     4966     Thr Leu Tyr Ile Pro Gln Gly Glu Ile Asp Gl - #y Leu Leu Thr Gly Gly     #   150     - ATA TGG GAG GCA ACA CTA GAG TTA CGA GTC AA - #A AGG CAT TAC GAC TAT     5014     Ile Trp Glu Ala Thr Leu Glu Leu Arg Val Ly - #s Arg His Tyr Asp Tyr     155                 1 - #60                 1 - #65                 1 -     #70     - AAT CAT GGT ACT TAC AAA GTT AAT ATC ACA GT - #T GAT TTG ACA GAC AAA     5062     Asn His Gly Thr Tyr Lys Val Asn Ile Thr Va - #l Asp Leu Thr Asp Lys     #               185     - GGA AAT ATT CAG GTC TGG ACA CCA AAG TTT CA - #T AGC GAT CCT AGA ATT     5110     Gly Asn Ile Gln Val Trp Thr Pro Lys Phe Hi - #s Ser Asp Pro Arg Ile     #           200     - GAT CTG AAT TTA CGT CCT GAA GGT AAT GGT AA - #A TAT TCT GGT AGT AAC     5158     Asp Leu Asn Leu Arg Pro Glu Gly Asn Gly Ly - #s Tyr Ser Gly Ser Asn     #       215     - GTG CTT GAG ATG TGT CTC TAT GAT GGC TAT AG - #T ACA CAT AGT CAA AGT     5206     Val Leu Glu Met Cys Leu Tyr Asp Gly Tyr Se - #r Thr His Ser Gln Ser     #   230     - ATA GAA ATG AGG TTT CAG GAT GAC TCA CAA AC - #A GGA AAT AAT GAA TAT     5254     Ile Glu Met Arg Phe Gln Asp Asp Ser Gln Th - #r Gly Asn Asn Glu Tyr     235                 2 - #40                 2 - #45                 2 -     #50     - AAT CTT ATA AAA ACT GGA GAG CCA TTA AAA AA - #A TTG CCA TAT AAA CTT     5302     Asn Leu Ile Lys Thr Gly Glu Pro Leu Lys Ly - #s Leu Pro Tyr Lys Leu     #               265     - TCT CTT CTT TTA GGA GGA CGA GAG TTT TAT CC - #A AAT AAT GGA GAG GCT     5350     Ser Leu Leu Leu Gly Gly Arg Glu Phe Tyr Pr - #o Asn Asn Gly Glu Ala     #           280     - TTT ACT ATT AAT GAT ACT TCG TCA TTG TTT AT - #A AAC TGG AAT CGT ATT     5398     Phe Thr Ile Asn Asp Thr Ser Ser Leu Phe Il - #e Asn Trp Asn Arg Ile     #       295     - AAG TCT GTA TCC TTA CCA CAG ATT AGT ATT CC - #A GTA CTA TGC TGG CCA     5446     Lys Ser Val Ser Leu Pro Gln Ile Ser Ile Pr - #o Val Leu Cys Trp Pro     #   310     - GCA AAC TTG ACA TTT ATG TCA GAG CTA AAT AA - #T CCA GAA GCG GGT GAG     5494     Ala Asn Leu Thr Phe Met Ser Glu Leu Asn As - #n Pro Glu Ala Gly Glu     315                 3 - #20                 3 - #25                 3 -     #30     - TAT TCA GGA ATA CTT AAC GTA ACA TTT ACT CC - #T AGT AGT TCA AGT CTG     5542     Tyr Ser Gly Ile Leu Asn Val Thr Phe Thr Pr - #o Ser Ser Ser Ser Leu     #               345     - TAA AAATAGTATC TTTATAAATT ATGCTATTTG CGGGAGACTT TATCAGCTG - #G     5595      *     - GAATTAGAGT CGCAATGATG TTTATCGGTA AACCAGCACC ATACTTCGGA AA - #ATGCTGGC     5655     - AAGCTTACGC CAATCTTTTT AGATTGAGTT GTTGGTATTA GATATCATAG TA - #AATGGTTA     5715     - GCTTGTAAAG TTAGCGCTAT CATGAAATAT TTGATTTTTA TAATATTAAA AG - #AGTCCCTC     5775     #              5798CCCA AAA     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:  238 ami - #no acids               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     - Met Lys Ile Leu Leu Phe Val Ile Leu Phe Ph - #e Asn Val Phe Ala Ala     - Ser Ala Asn Phe Met Val Tyr Pro Ile Ser Ly - #s Asp Ile Gln Ser Gly     #       10     - Gly Ser Glu Thr Ile Lys Val Phe Ser Lys Se - #r Lys Asp Val Gln Tyr     # 30     - Ile Lys Ile Tyr Thr Lys Arg Val Ile Asn Pr - #o Gly Thr Lys Glu Glu     #                 45     - Gln Glu Val Asp Ile Lys Asn Trp Asp Gly Gl - #y Leu Ile Val Thr Pro     #             60     - Ala Lys Val Val Leu Pro Ala Gly Ala Ser Ly - #s Ser Ile Arg Leu Thr     #         75     - Glu Ile Asn Lys Lys Glu Gln Glu Glu Val Ty - #r Arg Val Tyr Phe Glu     #     90     - Ser Val Lys Pro Gly Gln Gln Asp Asp Ile Gl - #u Glu Lys Asn Gly Arg     #110     - Val Asn Thr Asp Leu Ser Val Asn Ile Ile Ty - #r Ala Ala Leu Ile Arg     #               125     - Thr Ser Pro Glu Asn Pro Gln Arg Lys Leu As - #p Val Ser Ile Glu Ser     #           140     - Asn Asn Val Trp Ile Lys Asn Thr Gly Asn Il - #e Arg Leu Gly Ile Lys     #       155     - Asp Val Phe Leu Cys Asp Thr Thr Ser Ile As - #n Asp Lys Cys Ala Lys     #   170     - Phe Ser Tyr Asn Arg Asn Leu Tyr Pro Asp Me - #t Ser Val Asp Thr Lys     175                 1 - #80                 1 - #85                 1 -     #90     - Leu Gly Lys Lys Gly Phe Ser Tyr Ala Val Il - #e Asp Thr Lys Asp Asp     #               205     - Arg Asn Glu Asn Ser Gly Glu Leu Ile Asn Il - #e Lys Leu Pro     #           220     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:  170 ami - #no acids               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     - Met Lys Leu Asn Lys Ile Ile Gly Ala Leu Va - #l Leu Ser Ser Thr Phe     10     - Val Ser Met Gly Ala Ser Ala Ala Glu Lys As - #n Ile Thr Val Thr Ala     #       5  1     - Ser Val Asp Pro Thr Ile Asp Leu Met Gln Se - #r Asp Gly Thr Ala Leu     # 25     - Pro Ser Ala Val Asn Ile Ala Tyr Leu Pro Gl - #y Glu Lys Arg Phe Glu     #                 40     - Ser Ala Arg Ile Asn Thr Gln Val His Thr As - #n Asn Lys Thr Lys Gly     #             55     - Ile Gln Ile Lys Leu Thr Asn Asp Asn Val Va - #l Met Thr Asn Leu Ser     #         70     - Asp Pro Ser Lys Thr Ile Pro Leu Glu Val Se - #r Phe Ala Gly Thr Lys     #     85     - Leu Ser Thr Ala Ala Thr Ser Ile Thr Ala As - #p Gln Leu Asn Phe Gly     #105     - Ala Ala Gly Val Glu Thr Val Ser Ala Thr Ly - #s Glu Leu Val Ile Asn     #               120     - Ala Gly Ser Thr Gln Gln Thr Asn Ile Val Al - #a Gly Asn Tyr Gln Gly     #           135     - Leu Val Ser Ile Val Leu Thr Gln Glu Pro     #       145     - (2) INFORMATION FOR SEQ ID NO:4:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:  866 ami - #no acids               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     - Met Arg Ala Phe Asn Lys Ile Thr Val Phe Il - #e Leu Phe Ile Pro Gly     15     - Leu Cys Phe Gly Thr Asn Gly Leu Glu Ser Ly - #s Lys Asn Ile Pro Glu     # 1               5     - Glu Phe Ile Asp Leu Trp Met Glu Gln Asp Gl - #u Leu Leu Glu Val Asn     #             20     - Leu Tyr Gly Arg Ser Leu Gly Val His Arg Va - #l Leu Thr Thr Pro Thr     #         35     - Thr Val Lys Phe Ser Ser Val Glu Glu Ile Le - #u Glu Lys Ile Asn Val     #     50     - Lys Gln Glu Lys Lys Glu Asp Leu Arg Ser Le - #u Leu Leu Gln Ser Tyr     # 70     - Ser Arg Asn Gly Asn Met Ser Cys Asn Gly Ph - #e Asp Glu Lys Glu Tyr     #                 85     - Ser Cys Asn Tyr Ile Arg Thr Asp Thr Val As - #n Val Ile Val Asp Glu     #            100     - Glu Asn Asn Glu Leu Asn Leu Phe Ile Gly Al - #a Ser Phe Leu Ser Val     #       115     - Gln Ala Gln Asp Asn Ile Tyr Tyr Gln Lys As - #n Ile Asn Ser Glu Lys     #   130     - Ala Phe Ile His Ser Gln Thr Ile Asn Phe Se - #r Glu Ser Glu Gly Tyr     135                 1 - #40                 1 - #45                 1 -     #50     - Lys Ser Leu Ser Leu Lys Gly Val Gly Ala Gl - #n Gly Leu Thr Glu Asn     #               165     - Ser Tyr Leu Val Phe Gly Trp Asp Ala Ile Ty - #r Asn Ser Ser Arg Lys     #           180     - Tyr Thr Tyr Lys Asn Gln Ser Ile Asn Asn Il - #e Tyr Tyr Arg Tyr Asp     #       195     - Phe Asp Lys Lys Tyr Tyr Tyr Gln Leu Gly Ar - #g Met Asp Arg Ser Asp     #   210     - Leu Ser Ser Ala Ser Ser Gly Asn Phe Asn Ph - #e Asn Met Leu Pro Leu     215                 2 - #20                 2 - #25                 2 -     #30     - Pro Asp Ile Asp Gly Phe Gln Ile Gly Thr Th - #r Gln Ser Tyr Ile Lys     #               245     - Asn Ile Glu Lys Ser Ile Ser Ser Pro Val Th - #r Val Met Leu Thr Arg     #           260     - Phe Ser Arg Val Glu Ala Phe Arg Asn Glu Gl - #u Leu Leu Gly Val Trp     #       275     - Tyr Leu Asn Ser Gly Ile Asn Asp Leu Asp Th - #r Ser Arg Leu Pro Asp     #   290     - Gly Ser Tyr Asp Leu Thr Leu Lys Ile Phe Gl - #u Gln Asp Ile Leu Val     295                 3 - #00                 3 - #05                 3 -     #10     - Arg Glu Glu Lys Val Pro Phe Asn Lys Gly Gl - #y Ala Ser Phe Gly Asp     #               325     - Met Gln Trp Asp Val Phe Ala Gln Ala Gly As - #n Ile Val Asn Asn Asn     #           340     - Asp Ser Tyr Ile Glu Lys Gln Thr Asn Lys Ly - #s Thr Gly Ile Asn Ala     #       355     - Gly Ile Arg Thr Pro Val Thr Arg Asn Leu Se - #r Phe Leu Gln Gly Gly     #   370     - Ala Ile Ile Asp Asn Asp Lys Tyr Tyr Glu Al - #a Gly Val Asn Trp Arg     375                 3 - #80                 3 - #85                 3 -     #90     - Ser Gly Phe Leu Asp Gly Val Leu Ser Gly As - #n Phe Ser Phe Leu Tyr     #               405     - Gly Asp Gly Ala Arg Gly Asn Tyr Gln Asn Il - #e Ser Tyr Thr Asp Gly     #           420     - Phe Asn Leu Ser Phe Tyr Arg Asn Asp Lys Se - #r Val Asp Asn Cys Ser     #       435     - His Asn Tyr Ser Ala Gly Trp Ser Gly Cys Ty - #r Glu Ser Tyr Ser Phe     #   450     - Ser Leu Ser Val Pro Val Ser Gly Trp Thr Th - #r Thr Leu Gly Tyr Asn     455                 4 - #60                 4 - #65                 4 -     #70     - His Thr Asn Asn Glu Ala Val His Lys Tyr As - #p Tyr Thr Pro Glu Tyr     #               485     - Phe Phe Ser Lys Lys Tyr Lys Gly Val Ser Ly - #s Arg Trp Gln Leu Thr     #           500     - Ser Ser Ser Ser Tyr Lys Trp Met Asp Tyr Hi - #s Val Ile Pro Thr Ile     #       515     - Gly Val Tyr Arg Ser Asp Gln Ser Arg Trp Se - #r Glu Gln Gly Gly Tyr     #   530     - Phe Ser Leu Ser Phe Thr Arg Val Lys Glu As - #n Ser Ala Ile Asn Ala     535                 5 - #40                 5 - #45                 5 -     #50     - Gly Tyr Ser Tyr Asn Tyr Val Lys His Lys As - #n Ala Thr His Glu Ala     #               565     - Phe Leu Asp Gly Arg Ile Thr Thr Asn Thr Ph - #e Gly Tyr Ser Glu Leu     #           580     - Gly Ser Arg Ile Asn Thr Asn Lys Asn Asn Th - #r Glu Ala Gly Val Thr     #       595     - Gly Arg Val Lys Asn Arg Phe Gly Asp Leu As - #n Gly Ser Leu Asn Val     #   610     - Asn Lys Ser Lys Thr Ser Gly Lys Met Thr Hi - #s Ser Met Ser Ala Asn     615                 6 - #20                 6 - #25                 6 -     #30     - Tyr Asn Ser Ser Phe Ala Ile Thr Gly Asp Se - #r Val Tyr Trp Gly Gly     #               645     - Asp Ala Ser Gly Leu Thr Lys Leu Ser Gly Gl - #y Val Val Asn Val Arg     #           660     - Ser Asp Asp Lys Ser Lys Glu Leu Ile Lys Il - #e Ser Gly Ser Ser Tyr     #       675     - Gly Asn Tyr Ile Leu Gly Ser Asn Asp Arg Se - #r Phe Ile Pro Val Ser     #   690     - Ala Leu Met Pro Ser Asn Leu Thr Ile Glu Gl - #u Ile Gln Ser Asn Asp     695                 7 - #00                 7 - #05                 7 -     #10     - Lys Asn Ile Thr Val Gln Ala Leu Ser Lys As - #n Asp Phe Phe Ile Leu     #               725     - Pro Gly Asn Val Phe Pro Ile Asp Val Thr Al - #a Asn Val Thr Val Ser     #           740     - Tyr Ile Gly Arg Ala Leu Asp Asp Lys Gly As - #n Pro Leu Ser Asn Ala     #       755     - His Ile Leu Asp Val His Gly Val Arg Leu As - #p Glu Asp Gly Gly Phe     #   770     - Ser Phe Glu Thr Ser Ala Gln Lys Lys Ser Le - #u Phe Leu Leu Lys Asp     775                 7 - #80                 7 - #85                 7 -     #90     - Lys Asp Ile Tyr Ser Cys Asp Val Lys Lys Ty - #r Asp Leu Arg Ser Gly     #               805     - Val Leu Phe Thr Gly Asp Leu Ile Cys Glu Hi - #s Ser Gly Ile Glu Arg     #           820     - Leu Gly Lys Asp Leu Val Asn Asn Pro Arg Va - #l Lys Gln Leu Leu Ala     #       835     - Tyr Lys         840     - (2) INFORMATION FOR SEQ ID NO:5:     -      (i) SEQUENCE CHARACTERISTICS:               (A) LENGTH:  364 ami - #no acids               (B) TYPE: amino acid               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     - Leu Lys Lys Val Ile Phe Val Leu Ser Met Ph - #e Leu Cys Ser Gln Val     5     - Tyr Gly Gln Ser Trp His Thr Asn Val Glu Al - #a Gly Ser Ile Asn Lys     #       10     - Thr Glu Ser Ile Gly Pro Ile Asp Arg Ser Al - #a Ala Ala Ser Tyr Pro     # 30     - Ala His Tyr Ile Phe His Glu His Val Ala Gl - #y Tyr Asn Lys Asp His     #                 45     - Ser Leu Phe Asp Arg Met Thr Phe Leu Cys Me - #t Ser Ser Thr Asp Ala     #             60     - Ser Lys Gly Ala Cys Pro Thr Gly Glu Asn Se - #r Lys Ser Ser Gln Gly     #         75     - Glu Thr Asn Ile Lys Leu Ile Phe Thr Glu Ly - #s Lys Ser Leu Ala Arg     #     90     - Lys Thr Leu Asn Leu Lys Gly Tyr Lys Arg Ph - #e Leu Tyr Glu Ser Asp     #110     - Arg Cys Ile His Tyr Val Asp Lys Met Asn Le - #u Asn Ser His Thr Val     #               125     - Lys Cys Val Gly Ser Phe Thr Arg Gly Val As - #p Phe Thr Leu Tyr Ile     #           140     - Pro Gln Gly Glu Ile Asp Gly Leu Leu Thr Gl - #y Gly Ile Trp Glu Ala     #       155     - Thr Leu Glu Leu Arg Val Lys Arg His Tyr As - #p Tyr Asn His Gly Thr     #   170     - Tyr Lys Val Asn Ile Thr Val Asp Leu Thr As - #p Lys Gly Asn Ile Gln     175                 1 - #80                 1 - #85                 1 -     #90     - Val Trp Thr Pro Lys Phe His Ser Asp Pro Ar - #g Ile Asp Leu Asn Leu     #               205     - Arg Pro Glu Gly Asn Gly Lys Tyr Ser Gly Se - #r Asn Val Leu Glu Met     #           220     - Cys Leu Tyr Asp Gly Tyr Ser Thr His Ser Gl - #n Ser Ile Glu Met Arg     #       235     - Phe Gln Asp Asp Ser Gln Thr Gly Asn Asn Gl - #u Tyr Asn Leu Ile Lys     #   250     - Thr Gly Glu Pro Leu Lys Lys Leu Pro Tyr Ly - #s Leu Ser Leu Leu Leu     255                 2 - #60                 2 - #65                 2 -     #70     - Gly Gly Arg Glu Phe Tyr Pro Asn Asn Gly Gl - #u Ala Phe Thr Ile Asn     #               285     - Asp Thr Ser Ser Leu Phe Ile Asn Trp Asn Ar - #g Ile Lys Ser Val Ser     #           300     - Leu Pro Gln Ile Ser Ile Pro Val Leu Cys Tr - #p Pro Ala Asn Leu Thr     #       315     - Phe Met Ser Glu Leu Asn Asn Pro Glu Ala Gl - #y Glu Tyr Ser Gly Ile     #   330     - Leu Asn Val Thr Phe Thr Pro Ser Ser Ser Se - #r Leu     335                 3 - #40                 3 - #45     - (2) INFORMATION FOR SEQ ID NO:6:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 19 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: other nucleic acid     #= "Oligonucleotide primer forsc                    PCR."     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     # 19               CAC     - (2) INFORMATION FOR SEQ ID NO:7:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 19 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: other nucleic acid     #= "Oligonucleotide primer forsc                    PCR."     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     # 19               ATC     - (2) INFORMATION FOR SEQ ID NO:8:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 28 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: other nucleic acid     #= "Oligonucleotide primer forsc                    PCR."     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     #             28   AACG ACGGCCAG     - (2) INFORMATION FOR SEQ ID NO:9:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 35 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: other nucleic acid     #= "Oligonucleotide primer forsc                    PCR."     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     #       35         CCGC CAAGCTTGCA TGCCT     - (2) INFORMATION FOR SEQ ID NO:10:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 171 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     -      Met Lys Leu Lys Lys Thr Ile Gly - # Ala Met Ala Leu Ala Thr Leu     Phe     #   15     -      Ala Thr Met Gly Ala Ser Ala Val - # Glu Lys Thr Ile Ser Val Thr     Ala     #                 30     -      Ser Val Asp Pro Thr Val Asp Leu - # Leu Gln Ser Asp Gly Ser Ala     Leu     #             45     -      Pro Asn Ser Val Ala Leu Thr Tyr - # Ser Pro Ala Val Asn Asn Phe     Glu     #         60     -      Ala His Thr Ile Asn Thr Val Val - # His Thr Asn Asp Ser Asp Lys     Gly     #     80     -      Val Val Val Lys Leu Ser Ala Asp - # Pro Val Leu Ser Asn Val Leu     Asn     #   95     -      Pro Thr Leu Gln Ile Pro Val Ser - # Val Asn Phe Ala Gly Lys Pro     Leu     #                110     -      Ser Thr Thr Gly Ile Thr Ile Asp - # Ser Asn Asp Leu Asn Phe Ala     Ser     #            125     -      Ser Gly Val Asn Lys Val Ser Ser - # Thr Gln Lys Leu Ser Ile His     Ala     #        140     -      Asp Ala Thr Arg Val Thr Gly Gly - # Ala Leu Thr Ala Gly Gln Tyr     Gln     #    160     -      Gly Leu Val Ser Ile Ile Leu Thr - # Lys Ser Thr     #   170     - (2) INFORMATION FOR SEQ ID NO:11:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 170 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     -      Met Lys Phe Lys Lys Thr Ile Gly - # Ala Met Ala Leu Thr Thr Met     Phe     #   15     -      Val Ala Val Ser Ala Ser Ala Val - # Glu Lys Asn Ile Thr Val Thr     Ala     #                 30     -      Ser Val Asp Pro Ala Ile Asp Leu - # Leu Gln Ala Asp Gly Asn Ala     Leu     #             45     -      Pro Ser Ala Val Lys Leu Ala Tyr - # Ser Pro Ala Ser Lys Thr Phe     Glu     #         60     -      Ser Tyr Arg Val Met Thr Gln Val - # His Thr Asn Asp Ala Thr Lys     Lys     #     80     -      Val Ile Val Lys Leu Ala Asp Thr - # Pro Gln Leu Thr Asp Val Leu     Asn     #   95     -      Ser Thr Val Gln Met Pro Ile Ser - # Val Ser Trp Gly Gly Gln Val     Leu     #                110     -      Ser Thr Thr Ala Lys Glu Phe Glu - # Ala Ala Ala Leu Gly Tyr Ser     Ala     #            125     -      Ser Gly Val Asn Gly Val Ser Ser - # Ser Gln Glu Leu Val Ile Ser     Ala     #        140     -      Ala Pro Lys Thr Ala Gly Thr Ala - # Pro Thr Ala Gly Asn Tyr Ser     Gly     #    160     -      Val Val Ser Leu Val Met Thr Leu - # Gly Ser     #   170     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 238 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     -      Met Arg Lys Leu Phe Leu Ser Leu - # Leu Met Ile Pro Phe Val Ala     Lys     #   15     -      Ala Asn Phe Met Ile Tyr Pro Ile - # Ser Lys Glu Ile Lys Gly Gly     Ser     #                 30     -      Ser Glu Leu Ile Arg Ile Tyr Ser - # Lys Ser Lys Asp Thr Gln Tyr     Ile     #             45     -      Lys Val Tyr Thr Lys Lys Val Leu - # Asn Pro Gly Thr Lys Glu Glu     Tyr     #         60     -      Glu Val Asp Thr Pro Asn Trp Glu - # Gly Gly Leu Val Thr Thr Pro     Ser     #     80     -      Lys Val Ile Leu Pro Gly Gly Gly - # Ser Lys Ser Val Arg Leu Ser     Gln     #   95     -      Leu Lys Asp Ile Ser Ser Glu Asp - # Val Tyr Arg Val Tyr Phe Glu     Ser     #                110     -      Ile Lys Pro Glu Lys Gln Asp Gly - # Leu Ser Lys Asn Lys Ser Leu     Lys     #            125     -      Thr Asp Leu Ser Val Asn Ile Ile - # Tyr Ala Ala Leu Ile Arg Val     Leu     #        140     -      Pro Lys Asp Gly Lys Ser Asp Met - # Arg Ala Ser Leu Ser Pro Lys     Ser     #    160     -      Ser Leu Leu Ile Lys Asn Thr Gly - # Asn Val Arg Val Gly Ile Lys     Asp     #   175     -      Ala Phe Phe Cys Lys Lys Thr Ser - # Ile Asn Asn Asp Asp Cys Ile     Lys     #                190     -      Lys Thr Tyr Asn Lys Asn Ile Tyr - # Pro Gly Ser Ser Phe Asp Thr     Gly     #            205     -      Val Ile Gln Asn Gly Phe Ser His - # Ile Phe Ile Asp Ser Val Asp     Gly     #        220     -      Ser Ala Gly Lys Gln Gly Lys Arg - # Met Leu Ile Ser Ile His     #    235     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 238 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     -      Met His Lys Leu Phe Tyr Leu Leu - # Ser Leu Leu Met Ala Pro Phe     Val     #   15     -      Ala Asn Ala Asn Phe Met Ile Tyr - # Pro Ile Ser Lys Asp Leu Lys     Asn     #                 30     -      Gly Asn Ser Glu Leu Val Arg Val - # Tyr Ser Lys Ser Lys Glu Ile     Gln     #             45     -      Tyr Ile Lys Ile Tyr Thr Lys Lys - # Ile Ile Asn Pro Gly Thr Thr     Glu     #         60     -      Glu Tyr Lys Val Asp Ile Pro Asn - # Trp Asp Gly Gly Leu Val Val     Thr     #     80     -      Pro Gln Lys Val Ile Leu Pro Ala - # Gly Ala Ser Lys Ser Ile Arg     Leu     #   95     -      Thr Gln Phe Lys Ile Pro Lys Lys - # Glu Glu Val Tyr Arg Val Tyr     Phe     #                110     -      Glu Ala Val Lys Pro Asp Ser Lys - # Glu Asn Val Ile Asp Asn Lys     Lys     #            125     -      Leu Thr Thr Glu Leu Ser Val Asn - # Ile Ile Tyr Ala Ala Leu Ile     Arg     #        140     -      Ser Leu Pro Ser Glu Gln Asn Ile - # Ser Leu Asn Ile Ser Arg Asn     Ala     #    160     -      Lys Lys Asn Ile Ile Ile Tyr Asn - # Asn Gly Asn Val Arg Ala Gly     Val     #   175     -      Lys Asp Ile Tyr Phe Cys Lys Ser - # Ser Asn Ile Asp Asp Asn Cys     Val     #                190     -      Lys Lys Ala Tyr Asn Lys Asn Ile - # Tyr Pro Glu Lys Ser Phe Asp     Thr     #            205     -      Leu Val Asn Asn Asn Phe Ser Tyr - # Val Phe Ile Lys Leu Asn His     Glu     #        220     -      Gly Ile Glu Lys Glu Gln Gly Leu - # Ile Gln Leu Lys Val Pro     #    235     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 890 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     -      Cys Cys Met Ile Gly Gly Lys Ser - # Ser Lys Val Val Ile Val Leu     Ser     #   15     -      Val Leu Ile Gly Ser Ser Ser Gly - # Phe Ala Ser Lys Tyr Asn Leu     Val     #                 30     -      Asp Ile Pro Glu Ser Phe Arg Asp - # Leu Trp Gly Glu Gln Asp Glu     Leu     #             45     -      Leu Glu Val Arg Cys Cys Leu Tyr - # Gly Gln Ser Leu Gly Val His     Arg     #         60     -      Ile Lys Ser Thr Pro Thr Thr Val - # Ala Phe Glu Ser Pro Asp Asn     Leu     #     80     -      Leu Asp Lys Ile Glu Ile Asn Lys - # Gly Lys Glu Ala Asp Leu Arg     Val     #   95     -      Leu Met Arg Gly Ser Phe Gln Arg - # Cys Cys Asn Gly Asn Met Ser     Cys     #                110     -      Gln Gly Tyr Thr Gly Gln Asn Asn - # Cys Asn Tyr Ile Lys Thr Asn     Thr     #            125     -      Val Ala Val Ile Val Asp Asp Val - # Glu Asn Val Leu Asn Leu Phe     Ile     #        140     -      Gly Asn Glu Phe Leu Ala Ser Gly - # Glu Asn Asp Cys Cys Ser Asp     Tyr     #    160     -      Tyr Gln Pro Ser Lys Asn Thr Lys - # Lys Ala Phe Ile His Ser Gln     Thr     #   175     -      Ile Asn Leu Ser Asp Thr Gly Asn - # Tyr Glu Asn Leu Ser Ile Val     Gly     #                190     -      Thr Gly Ser Leu Gly Ile Thr Asp - # Asn Ser Tyr Ala Ile Leu Gly     Cys     #            205     -      Cys Trp Ala Ala Asn Tyr Asn Arg - # Tyr Lys Ser Tyr Asn Tyr Asn     Glu     #        220     -      Gln Ser Ile Asn Ser Leu Tyr Phe - # Arg His Asp Phe Glu Lys Asn     Phe     #    240     -      Tyr Tyr Gln Leu Gly Arg Ile Asp - # Arg Ser Asp Leu Ser Gln Ser     Ser     #   255     -      Gly Gly Asn Cys Cys Phe Asn Phe - # Asp Leu Leu Pro Val Pro Asp     Ile     #                270     -      Tyr Gly Met Arg Ala Gly Thr Thr - # Gln Ser Tyr Ile Lys Asn Thr     Gly     #            285     -      Lys Ser Val Ala Ser Pro Val Thr - # Ile Met Leu Thr His Phe Ser     Arg     #        300     -      Val Glu Ala Tyr Arg Asn Gly Cys - # Cys Gln Leu Leu Gly Val Trp     Tyr     #    320     -      Leu Asp Ala Gly Ile Ser Glu Leu - # Asp Thr Glu Arg Leu Pro Asp     Gly     #   335     -      Asn Tyr Asp Leu Lys Leu Lys Ile - # Phe Glu Gln Glu Gln Leu Val     Arg     #                350     -      Glu Glu Ile Val Pro Phe Asn Lys - # Ser Gly Ser Cys Cys Ser Ile     Gly     #            365     -      Asp Thr His Trp Asp Val Phe Val - # Gln Ala Gly Asp Ile Ile Asn     Asp     #        380     -      Asn Gly Arg Tyr Val Glu Lys Gln - # Lys Asn His Lys Ser Ala Ile     Asn     #    400     -      Ser Gly Leu Arg Leu Pro Leu Thr - # Arg Asn Leu Ala Val Gln Leu     Cys     #   415     -      Cys Gly Gly Ala Val Ile Asp Asn - # Lys Asn Tyr Tyr Glu Thr Gly     Ile     #                430     -      Leu Trp Asn Ser Gly Leu Leu Asp - # Gly Ser Leu Asn Ser Lys Phe     Thr     #            445     -      Phe Leu Phe Gly Asp Asp Thr His - # Gly Asn Tyr Gln Asn Val Ser     Tyr     #        460     -      Thr Asp Gly Phe Ser Leu Ser Phe - # Tyr His Asn Asp Lys Arg Val     Asp     #    480     -      Asp Cys Gly Lys Asp Tyr Asn Met - # Gly Trp Ser Gly Cys Tyr Glu     Ser     #   495     -      Tyr Ser Ala Ser Leu Ser Ile Pro - # Val Lys Gly Trp Asn Ser Thr     Leu     #                510     -      Ala Tyr Ser Asn Thr Tyr Ser Thr - # Ser Val Tyr Arg Tyr Asp Ala     Val     #            525     -      Ser Glu Tyr Val Pro Tyr Tyr Tyr - # Tyr Lys Gly Arg Thr Lys Arg     Trp     #        540     -      Gln Leu Thr Ala Ser Thr Val Val - # Arg Trp Gly Asp Tyr Asn Ile     Met     #    560     -      Pro Thr Ile Gly Val Tyr Asn Ser - # Glu Gln Lys Gln Trp Ala Asp     Lys     #   575     -      Gly Gly Tyr Leu Ser Leu Thr Leu - # Thr Arg Val Asp Gly Gly Lys     Ser     #                590     -      Leu Asn Ala Gly Tyr Ser Tyr Asn - # Tyr Ser Arg Gly Asn Tyr Thr     Ser     #            605     -      Asn Asp Ala Phe Val Glu Gly His - # Leu Val Ser Asp Thr Asn Val     Ser     #        620     -      Tyr Arg Glu Leu Ser Ala Arg Val - # Ser Gly Asn Arg Tyr Tyr Thr     Glu     #    640     -      Gly Gly Val Ser Gly Arg Ile Asn - # Asn Arg Phe Gly Asp Leu Asn     Gly     #   655     -      Thr Leu Asn Val Asn Lys Asn Arg - # Lys Ser His Asp Thr Thr His     Ser     #                670     -      Leu Thr Ala Gly Tyr Ser Ser Ser - # Phe Ala Leu Thr Thr Asp Gly     Ile     #            685     -      Tyr Trp Gly Gly Ser Ala Ser Gly - # Leu Thr Asn Leu Ser Gly Gly     Ile     #        700     -      Val Arg Val Lys Ser Asn Glu Asp - # Glu Ser Glu Leu Leu Asn Val     Lys     #    720     -      Gly Ser Ser Tyr Gly His Tyr Ser - # Leu Gly Ser Asn Asp Ser Leu     Phe     #   735     -      Ile Pro Val Pro Ala Leu Met Gln - # Ala Ser Leu Thr Ile Glu Glu     Asn     #                750     -      Thr Asn Lys Ser Lys Asn Ile Asp - # Val Leu Ala Pro Thr Lys Asn     Thr     #            765     -      Phe Phe Met Leu Pro Gly Ser Val - # Tyr Pro Ile Asp Val Ser Ala     Asn     #        780     -      Val Ser Phe Thr Tyr Val Gly Arg - # Gly Val Asp Val Lys Gly Arg     Pro     #    800     -      Leu Ser Gly Ala Tyr Ile Leu Asn - # Ala Gln Asn Ile Val Leu Asp     Glu     #   815     -      Asn Gly Gly Phe Ser Phe Glu Ser - # Ser Glu Asn Glu Lys Glu Leu     Phe     #                830     -      Leu Leu Lys Asp Lys Thr Ile Tyr - # Ser Cys Ser Leu Asp Arg Ser     Glu     #            845     -      Met Arg Asn Gly Ile Ala Phe Val - # Gly Glu Val Ala Cys Asn Ser     Thr     #        860     -      Ile Lys Glu Leu Leu Pro Glu Lys - # Leu Val Thr Asn Ser Arg Ile     His     #    880     -      Asp Leu Leu Ala Tyr Asn Gln Asp - # Thr Glu     #   890     - (2) INFORMATION FOR SEQ ID NO:15:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 869 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     -      Met Lys His Lys Lys Lys Asn Arg - # Leu Val Val Ala Ile Ser Val     Ala     #   15     -      Leu Ile Pro Tyr Ile Gly Val Thr - # Gly Asp Ile Pro Asp Ser Phe     Arg     #                 30     -      Asp Leu Trp Gly Glu Gln Asp Glu - # Phe Tyr Glu Val Lys Leu Tyr     Gly     #             45     -      Gln Thr Leu Gly Ile His Arg Ile - # Lys Thr Thr Pro Thr His Ile     Lys     #         60     -      Phe Tyr Ser Pro Glu Ser Ile Leu - # Asp Lys Ile Asn Leu Lys Lys     Glu     #     80     -      Lys Glu Lys Glu Leu Ser Val Phe - # Phe Thr Asn Ser Phe Ser Arg     Asn     #   95     -      Gly Asn Met Ser Cys Gln Gly Asn - # Thr Thr Ile Gln Tyr Asn Cys     Asn     #                110     -      Tyr Ile Lys Thr Lys Ser Val Asp - # Val Ile Val Asp Asp Val Asp     Asn     #            125     -      Val Val Asn Leu Phe Ile Gly Asn - # Glu Phe Leu Asp Ser Glu Ala     His     #        140     -      Asn Asp Glu Tyr His Gln Leu Ser - # Arg Asn Val Lys Lys Ala Phe     Ile     #    160     -      Gln Ser Gln Thr Ile Asn Leu Ser - # Asp Ser Gly Lys Tyr Lys Arg     Leu     #   175     -      Ser Ile Ser Gly Asn Ser Ala Leu - # Gly Ile Thr Asp Thr Ser Tyr     Ala     #                190     -      Val Leu Asn Trp Trp Met Asn Tyr - # Asn Lys Ser Asn Gly Tyr Ser     Asn     #            205     -      Asn Glu Lys Thr Ile Asn Ser Leu - # Tyr Phe Arg His Asp Leu Asp     Lys     #        220     -      Arg Tyr Tyr Tyr Gln Phe Gly Arg - # Met Asp Arg Thr Asp Leu Ser     Gln     #    240     -      Ser Ile Ser Gly Ser Phe Asn Phe - # Asn Leu Leu Pro Leu Pro Asp     Ile     #   255     -      Asp Gly Ile Arg Thr Gly Thr Thr - # Gln Ser Tyr Ile Lys Asn Thr     Asp     #                270     -      Lys Phe Ile Ala Ser Pro Val Thr - # Val Met Leu Thr Asn Phe Ser     Arg     #            285     -      Val Glu Ala Phe Arg Asn Asp Gln - # Leu Leu Gly Val Trp Tyr Leu     Asp     #        300     -      Ser Gly Val Asn Glu Leu Asp Thr - # Ala Arg Leu Pro Tyr Gly Ser     Tyr     #    320     -      Asp Leu Lys Leu Lys Ile Phe Glu - # Asn Thr Gln Leu Val Arg Glu     Glu     #   335     -      Ile Ile Pro Phe Asn Lys Gly Arg - # Ser Ser Ile Gly Asp Met Gln     Trp     #                350     -      Asp Ile Phe Val Gln Gly Gly Asn - # Ile Val Asn Asp Asn Asp Arg     Tyr     #            365     -      Ile Glu Lys Gln Asn Asn His Lys - # Ser Ser Ile Asn Thr Gly Leu     Arg     #        380     -      Leu Pro Ile Thr Lys Asn Ile Ser - # Val Gln Gln Gly Val Ser Val     Ile     #    400     -      Asp Asn Lys Ser Tyr Tyr Glu Gly - # Ser Leu Lys Trp Asn Ser Gly     Ile     #   415     -      Leu Ser Gly Ser Leu Asn Ser Glu - # Phe Ser Phe Leu Trp Gly Asp     Asn     #                430     -      Ala Lys Gly Asn Tyr Gln Ser Ile - # Ser Tyr Thr Asp Gly Phe Ser     Leu     #            445     -      Ser Phe Tyr His Asn Asp Lys Arg - # Val Asp Asn Cys Gly Arg Asn     Tyr     #        460     -      Asn Ala Gly Trp Ser Gly Cys Tyr - # Glu Ser Tyr Ser Ala Ser Leu     Ser     #    480     -      Ile Pro Leu Leu Gly Trp Thr Ser - # Thr Leu Gly Tyr Ser Asp Thr     Tyr     #   495     -      Ser Glu Ser Val Tyr Lys Ser His - # Ile Leu Ser Glu Tyr Gly Phe     Tyr     #                510     -      Asn Gln Asn Ile Tyr Lys Gly Arg - # Thr Gln Arg Trp Gln Leu Thr     Ser     #            525     -      Ser Thr Ser Leu Lys Trp Met Asp - # Tyr Asn Phe Met Pro Ala Ile     Gly     #        540     -      Ile Tyr Asn Ser Glu Gln Arg Gln - # Leu Thr Asp Lys Gly Gly Tyr     Ile     #    560     -      Ser Val Thr Ile Thr Arg Ala Ser - # Arg Glu Asn Ser Leu Asn Thr     Gly     #   575     -      Tyr Ser Tyr Asn Tyr Ser Arg Gly - # Asn Tyr Ser Ser Asn Glu Leu     Phe     #                590     -      Val Asp Gly Tyr Met Thr Ser Thr - # Asn Asn Gly Asp Tyr His Glu     Ala     #            605     -      Gly Met Arg Phe Asn Lys Asn Arg - # His Asn Ala Glu Gly Arg Leu     Ser     #        620     -      Gly Arg Ile Asn Asn Arg Phe Gly - # Asp Leu Asn Gly Ser Phe Ser     Met     #    640     -      Asn Lys Asn Arg Asn Thr Asn Ser - # Thr Asn His Ser Leu Thr Gly     Gly     #   655     -      Tyr Asn Ser Ser Phe Ala Leu Thr - # Ser Asp Gly Phe Tyr Trp Gly     Gly     #                670     -      Ser Thr Ala Gly Leu Thr Lys Leu - # Ala Gly Gly Ile Ile Lys Val     Lys     #            685     -      Ser Asn Asp Thr Lys Lys Asn Leu - # Val Lys Val Thr Gly Thr Leu     Tyr     #        700     -      Gly Asp Tyr Ser Leu Gly Ser Asn - # Asp Asn Ala Phe Ile Pro Val     Pro     #    720     -      Ala Leu Thr Pro Ala Ser Leu Ile - # Ile Glu Asp Asn Asn Tyr Gly     Asp     #   735     -      Asn Asn Ile Ser Ile Leu Ala Pro - # Thr Asn Asn Asp Met Phe Met     Leu     #                750     -      Pro Gly Asn Val Tyr Pro Val Glu - # Ile Glu Thr Lys Val Ser Val     Ser     #            765     -      Tyr Ile Gly Arg Gly Phe Asp Pro - # Asn Gly Thr Pro Leu Ser Gly     Ala     #        780     -      His Val Leu Asn Glu Pro His Val - # Ile Leu Asp Glu Asp Gly Gly     Phe     #    800     -      Ser Phe Glu Tyr Thr Gly Asn Glu - # Lys Thr Leu Phe Leu Leu Lys     Gly     #   815     -      Arg Thr Ile Tyr Thr Cys Gln Leu - # Gly Lys Asn Lys Val His Lys     Gly     #                830     -      Ile Val Phe Val Gly Asp Val Ile - # Cys Asp Ile Asn Ser Thr Ser     Ser     #            845     -      Leu Pro Asp Glu Phe Val Lys Asn - # Pro Arg Val Gln Asp Leu Leu     Ala     #        860     -      Lys Asn Asp Lys Gly          865     - (2) INFORMATION FOR SEQ ID NO:16:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 363 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     -      Met Lys Lys Ile Phe Ile Phe Leu - # Ser Ile Ile Phe Ser Ala Val     Val     #   15     -      Ser Ala Gly Arg Tyr Pro Glu Thr - # Thr Val Gly Asn Leu Thr Lys     Ser     #                 30     -      Phe Gln Ala Pro Arg Leu Asp Arg - # Ser Val Gln Ser Pro Ile Tyr     Asn     #             45     -      Ile Phe Thr Asn His Val Ala Gly - # Tyr Ser Leu Ser His Ser Leu     Tyr     #         60     -      Asp Arg Ile Val Phe Leu Cys Thr - # Ser Ser Ser Asn Pro Val Asn     Gly     #     80     -      Ala Cys Pro Thr Ile Gly Thr Ser - # Gly Val Gln Tyr Gly Thr Thr     Thr     #   95     -      Ile Thr Leu Gln Phe Thr Glu Lys - # Arg Ser Leu Ile Lys Arg Asn     Ile     #                110     -      Asn Leu Ala Gly Asn Lys Lys Pro - # Ile Trp Glu Asn Gln Ser Cys     Asp     #            125     -      Phe Ser Asn Leu Met Val Leu Asn - # Ser Lys Ser Trp Ser Cys Gly     Ala     #        140     -      His Gly Asn Ala Asn Gly Thr Leu - # Leu Asn Leu Tyr Ile Pro Ala     Gly     #    160     -      Glu Ile Asn Lys Leu Pro Phe Gly - # Gly Ile Trp Glu Ala Thr Leu     Ile     #   175     -      Leu Arg Leu Ser Arg Tyr Gly Glu - # Val Ser Ser Thr His Tyr Gly     Asn     #                190     -      Tyr Thr Val Asn Ile Thr Val Asp - # Leu Thr Asp Lys Gly Asn Ile     Gln     #            205     -      Val Trp Leu Pro Gly Phe His Ser - # Asn Pro Arg Val Asp Leu Asn     Leu     #        220     -      Arg Pro Ile Gly Asn Tyr Lys Tyr - # Ser Gly Ser Asn Ser Leu Asp     Met     #    240     -      Cys Phe Tyr Asp Gly Tyr Ser Thr - # Asn Ser Asp Ser Met Val Ile     Lys     #   255     -      Phe Gln Asp Asp Asn Pro Thr Asn - # Ser Ser Glu Tyr Asn Leu Tyr     Lys     #                270     -      Ile Gly Gly Thr Glu Lys Leu Pro - # Tyr Ala Val Ser Leu Leu Met     Gly     #            285     -      Glu Lys Ile Phe Tyr Pro Val Asn - # Gly Gln Ser Phe Thr Ile Asn     Asp     #        300     -      Ser Ser Val Leu Glu Thr Asn Trp - # Asn Arg Val Thr Ala Val Ala     Met     #    320     -      Pro Glu Val Asn Val Pro Val Leu - # Cys Trp Pro Ala Arg Leu Leu     Leu     #   335     -      Asn Ala Asp Val Asn Asn Pro Glu - # Ala Gly Gln Tyr Met Gly Asn     Ile     #                350     -      Lys Ile Thr Phe Thr Pro Ser Ser - # Gln Thr Leu     #            360     - (2) INFORMATION FOR SEQ ID NO:17:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 360 amino               (B) TYPE: amino acid               (C) STRANDEDNESS:               (D) TOPOLOGY: unknown     -     (ii) MOLECULE TYPE: protein     -    (iii) HYPOTHETICAL: NO     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     -      Met Asn Lys Ile Leu Phe Ile Phe - # Thr Leu Phe Phe Ser Ser Gly     Phe     #   15     -      Phe Thr Phe Ala Val Ser Ala Asp - # Lys Asn Pro Gly Ser Glu Asn     Met     #                 30     -      Thr Asn Thr Ile Gly Pro His Asp - # Arg Gly Gly Ser Ser Pro Ile     Tyr     #             45     -      Asn Ile Leu Asn Ser Tyr Leu Thr - # Ala Tyr Asn Gly Ser His His     Leu     #         60     -      Tyr Asp Arg Met Ser Phe Leu Cys - # Leu Ser Ser Gln Asn Thr Leu     Asn     #     80     -      Gly Ala Cys Pro Ser Ser Asp Ala - # Pro Gly Thr Ala Thr Ile Asp     Gly     #   95     -      Glu Thr Asn Ile Thr Leu Gln Phe - # Thr Glu Lys Arg Ser Leu Ile     Lys     #                110     -      Arg Glu Leu Gln Ile Lys Gly Tyr - # Lys Gln Phe Leu Phe Lys Asn     Ala     #            125     -      Asn Cys Pro Ser Lys Leu Ala Leu - # Asn Ser Ser His Phe Gln Cys     Asn     #        140     -      Arg Glu Gln Ala Ser Gly Ala Thr - # Leu Ser Leu Tyr Ile Pro Ala     Gly     #    160     -      Glu Leu Asn Lys Leu Pro Phe Gly - # Gly Val Trp Asn Ala Val Leu     Lys     #   175     -      Leu Asn Val Lys Arg Arg Tyr Asp - # Thr Thr Tyr Gly Thr Tyr Thr     Ile     #                190     -      Asn Ile Thr Val Asn Leu Thr Asp - # Lys Gly Asn Ile Gln Ile Trp     Leu     #            205     -      Pro Gln Phe Lys Ser Asn Ala Arg - # Val Asp Leu Asn Leu Arg Pro     Thr     #        220     -      Gly Gly Gly Thr Tyr Ile Gly Arg - # Asn Ser Val Asp Met Cys Phe     Tyr     #    240     -      Asp Gly Tyr Ser Thr Asn Ser Ser - # Ser Leu Glu Ile Arg Phe Gln     Asp     #   255     -      Asp Asn Ser Lys Ser Asp Gly Lys - # Phe Tyr Leu Lys Lys Ile Asn     Asp     #                270     -      Asp Ser Lys Glu Leu Val Tyr Thr - # Leu Ser Leu Leu Leu Ala Gly     Lys     #            285     -      Asn Leu Thr Pro Thr Asn Gly Gln - # Ala Leu Asn Ile Asn Thr Ala     Ser     #        300     -      Leu Glu Thr Asn Trp Asn Arg Ile - # Thr Ala Val Thr Met Pro Glu     Ile     #    320     -      Ser Val Pro Val Leu Cys Trp Pro - # Gly Arg Leu Gln Leu Asp Ala     Lys     #   335     -      Val Lys Asn Pro Glu Ala Gly Glu - # Tyr Ser Gly Ile Leu Asn Val     Thr     #                350     -      Phe Thr Pro Ser Ser Ser Ser Leu     #            360     __________________________________________________________________________ 

We claim:
 1. An isolated non-naturally occurring recombinant nucleic acid molecule comprising a sequence encoding CS2 pilin protein having an amino acid sequence as given in SEQ ID NO:3.
 2. The nucleic acid molecule of claim 1 wherein said CS2 pilin-encoding sequence is as given in SEQ ID NO:1 from nucleotide 1255 through nucleotide
 1764. 3. The nucleic acid molecule of claim 1 further comprising sequences encoding proteins having amino acid sequences as given in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5.
 4. The nucleic acid molecule of claim 3 wherein said further coding sequences are as given in SEQ ID NO:1 from nucleotide 499 to nucleotide 1212, from nucleotide 1836 to nucleotide 4433 and from nucleotide 4451 to nucleotide
 5542. 5. The nucleic acid molecule of claim 3 wherein said molecule comprises a nucleotide sequence as given in SEQ ID NO:1, from nucleotide 499 to nucleotide
 5698. 6. An enteric bacterial cell into which the non-naturally occurring nucleic acid molecule of claim 1 has been introduced.
 7. An immunogenic composition comprising a DNA vaccine molecule capable of expressing at least one CS2-derived coding sequence in a human cell, wherein CS2-derived coding sequences encode proteins having amino acid sequences as given in SEQ ID NO:2: SEQ ID NO:3, amino acids 1 to 147: SEQ ID NO:4, amino acids 1 to 840: and SEQ ID NO:5.
 8. The immunogenic composition of claim 7 wherein said vaccine DNA molecule is capable of expressing a protein with an amino acid sequence as given in SEQ ID NO:3 from amino acid 1 to amino acid
 147. 9. The immunogenic composition of claim 7 wherein said vaccine DNA molecule is capable of expressing a protein with an amino acid sequence as given in SEQ ID NO:2 from amino acid 1 to amino acid
 220. 10. The immunogenic composition of claim 7 wherein said vaccine DNA molecule is capable of expressing a protein with an amino acid sequence as given in SEQ ID NO:4 from amino acid 1 to amino acid
 840. 11. The immunogenic composition of claim 7 wherein said vaccine DNA molecule is capable of expressing a protein with an amino acid sequence as given in SEQ ID NO:5 from amino acid 1 to amino acid
 346. 12. An enteric bacterial cell into which the non-naturally occurring nucleic acid molecule of claim 3 has been introduced. 